Recombinant multivalent viral vaccine

ABSTRACT

The present invention relates to multivalent recombinant raccoon poxviruses, containing more than one exogenous gene inserted into either the thymidine kinase gene, the hemagglutinin gene, or a combination thereof. Disclosed is the use of the multivalent recombinant raccoon poxviruses as vaccines to immunize felines against subsequent challenge by feline pathogens. Also disclosed is a method of making a a multivalent recombinant raccoon poxvirus by a recombination process involving the construction of an insertion vector into which the exogenous genes are inserted, and flanking the inserted genes are sequences which can recombine into the raccoon poxvirus thymidine kinase gene, or the hemagglutinin gene, or a combination thereof; introducing both the insertion vector containing the exogenous genes, and raccoon poxvirus into susceptible host cells; and selecting the recombinant raccoon poxvirus from the resultant plaques.

This application is a continuation-in-part of our earlier applicationU.S. Ser. No. 08/190,789, filed Jan. 27, 1994, now abandoned, which is acontinuation of our earlier application U.S. Ser. No. 07,726,609, filedJul. 9, 1991, now abandoned, which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the production and use of arecombinant viral vector as a multivalent vaccine in the protection offelines against infection by various viral pathogens of felines. Moreparticularly, the present invention relates to a recombinant multivalentvaccine formed by inserting multiple genes such as a felinepanleukopenia virus (FPV) gene, a rabies virus gene, and/or a felinecalicivirus (FCV) capsid protein gene each operably linked to apromoter, into a raccoon poxvirus (RCNV) for expression.

BACKGROUND OF THE INVENTION

1. Feline Panleukopenia Virus Biology and Pathology

Feline panleukopenia virus (FPV) is a highly contagious viral disease ofdomestic cats and exotic cats. The virus is transmittable to susceptiblecats by contact with body secretions and excretions of infected catsduring the acute phase of infection in which virus is shed. Aerosoltransmission, and transmission by insect vectors can also occur. Thevirus infects and destroys actively replicating cells in lymph nodes,and hematopoietic and gastrointestinal tissues of felines therebycausing sudden onset of symptoms including fever, anorexia, leukopenia,vomiting and diarrhea. In particular, FPV causes severe clinical illnessin young kittens with high morbidity and mortality. A marked drop intotal leukocyte count by day 4 to 6 after infection is the prominentindicator of FPV infection. Disease caused by infection with FPV hasbeen described as feline parvovirus, feline panleukopenia, infectiousenteritis, viral enteritis, cat “distemper”, granulocytosis, cat plague,and cat fever.

FPV is a small, single stranded DNA virus, a parvovirus, that requiresrapidly multiplying cells for DNA replication. The genome of FPV is alinear, single stranded DNA of about 5 kilobases in size that encodesthree structural proteins: a large 80-85 kilodalton (kd) protein (“VP1”)comprising 10% to 15% of the viral protein; a medium size protein of64-67 kd (“VP2”); and a part of the VP2 protein which is converted to a60-64 kd protein (“VP3”) by proteolytic cleavage. The three proteinsphysically form a nested set of proteins within which the viral DNA isenclosed. FPV is very closely related to canine parvovirus (CPV) andmink enteritis virus both on the protein and amino acid level (Tratschinet al., 1982, J. Gen. Virol., 61:33-41; Truyen et al., 1994, Virology200:494-503; and Truyen et al., 1994, J. Virol. 66:5399-5408). The highcross-reactivity between CPV and FPV indicated their antigenicsimilarities and the possibility of mutual neutralization and protectionbetween the two viruses.

2. Rabies Virus Biology and Pathology

Rabies virus is a member of the genus Lyssavirus in the familyRhabdoviridae, and contains an unsegmented negative stranded RNA genome.Of the five known viral structural proteins, the rabies virustransmembrane glycoprotein G plays a critical role for the induction andbinding of the virus-neutralization antibodies and the stimulation of Tcell-mediated immunity (Lafon et al., 1983, J. Gen. Virol. 64:843-851;Lafon et al., 1985, J. Gen. Virol. 66:2125-2133; Wiktor et al., 1973, J.Immunol. 110:269-276; Wiktor et al., 1984, Dev. Biol. Stand. 57:199-211;Wiktor et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7194-7198). Also,the arginine, at position 333 in the glycoprotein amino acid sequence,is essential for the integrity of at least one antigenic determinant andfor the ability of rabies virus to produce a lethal infection in adultmice (Dietzschold et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:70-74).Initial symptoms of rabies virus infection include fever, and malaise.The disease progresses rapidly to symptoms including agitation,convulsions, and coma; and eventually, if untreated, the infected animalmay die from organ failure.

3. Recombinant Veterinary Vaccines

In the art of veterinary vaccines, purified recombinant VP2 protein hasbeen used as an immunogen for protecting dogs against infection bycanine parvovirus (Wood et al., U.S. Pat. No. 4,971,793). In terms ofviral vaccine vectors, vaccinia virus recombinants have been constructedwith insertion of a respective gene encoding either feline infectiousperitonitis virus (FIPV) spike protein, membrane glycoprotein, ornucleocapsid protein (Vennema et al., 1990, J. Virol. 64:1407-1409;Vennema et al., 1991, Virology 181:327-335). Immunization with such avaccinia virus recombinant appeared to be of little or no value in theprotection of vaccinated kittens against challenge with FIPV. Thus,vaccinia virus vectors do not appear to be a good choice forconstructing feline recombinant vaccines because: (a) vacciniavirus/viral vectors did not elicit protection or detectable virusneutralization antibodies (Vennema et al., 1991, supra; Scott, 1988,Conf. Res. Workers Anim. Dis. 69:60); and (b) of concerns of introducingrecombinant vaccinia virus for veterinary or human use, particularlybecause of rare side effects associated with vaccinia virusimmunization.

In contrast, a raccoon poxvirus (RCNV) recombinant vector containing thegene encoding rabies virus surface glycoprotein G, has been usedsuccessfully to induce immunity in raccoons which is protective againstsubsequent challenge with raccoon rabies virus (Esposito et al., 1988,Virology, 165:313-316). However, the investigators report that thecomplete host range of RCNV is not known. A recombinant RCNV containingthe gene encoding FPV VP2 protein (“recombinant RCNV/FPV”) was recentlyconstructed by inserting the VP2 protein gene into a vaccinia expressionvector, and then recombining the insertion into the thymidine kinase(TK) gene of RCNV. In a vaccine trial, all cats immunized with theRCNV/FPV were fully protected against subsequent FPV challenge, andshowed high titers of FPV viral neutralization antibody (U.S. patentapplication Ser. No. 08/190,789 assigned to the assignee of the presentinvention). Presently, there are no reports of the design of afunctional multivalent RCNV-vectored vaccine, and its use for inducingprotective immunity in felines.

SUMMARY OF THE INVENTION

A novel raccoon poxvirus (RCNV) recombinant vaccine against both felinepanleukopenia virus (FPV) and rabies virus was developed based on thehomologous thymidine kinase gene between vaccinia virus and RCNV. Theinfectious recombinant virus (RCNV/FPV/RAB) carried both FPV VP2 andrabies G protein genes, each operably linked to a promoter. Vaccinetrials using RCNV/FPV/RAB induced strong immune responses in cats toboth FPV and rabies virus. Cats immunized with RCNV/FPV/RAB were fullyprotected against subsequent FPV challenge. Viral neutralizationantibody titers for both FPV and rabies virus were sufficient to protectcats from the related virulent virus infection or challenge. In anotherembodiment, a novel raccoon poxvirus multivalent recombinant vaccine canbe developed based on an insertion vector being constructed to havehomologous hemagglutinin (HA) gene sequences which flank one or moreinserted genes (such as FCV capsid protein gene), and thus allowingrecombination into the hemagglutinin gene sequences of raccoon poxvirus.Thus, the methods and compositions of the present invention provide thebasis for producing novel multivalent recombinant vaccine vectors forfelines. Using the methods according to the present invention, amultivalent recombinant raccoon poxvirus could have the followinginsertions: one or more exogenous genes recombined into both the raccoonpoxvirus TK and HA gene sequences; more than one exogenous generecombined into the raccoon poxvirus TK gene sequences; and more thanone exogenous gene recombined into the raccoon poxvirus HA genesequences.

Accordingly, it is one object of the present invention to provide amethod for inserting more than one exogenous gene into the thymidinekinase gene region and/or hemagglutinin gene region of raccoon poxvirusfor expression.

It is another object of the present invention to provide a method forinserting multiple genes encoding antigens of feline pathogens into thethymidine kinase gene region and/or hemagglutinin gene region of raccoonpoxvirus for expression.

It is a further object of the invention to provide vaccine compositions,comprising recombinant raccoon poxvirus, for eliciting a protectiveimmune response, to more than one feline pathogen, in felines receivingthe vaccine compositions.

These and other objects of the present invention will become readilyapparent from the ensuing description, embodiments and illustrations.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the insertion of a rabies-G protein gene,operably linked to a vaccinia virus P₁₁ late promoter, into arecombinant vaccinia virus recombinant plasmid carrying the FPV VP2gene, operably linked to a vaccinia virus P₁₁, late promoter, in forminga novel plasmid termed F3S/FPV/RAB.

FIG. 2 is a representation (magnification at 250×) showing indirectimmunofluorescent assay-positive plaques/cells infected with themultivalent recombinant raccoon poxvirus. Specific fluorescence,indicating expression of VP2, was concentrated in the cytoplasm ofinfected cells, and was also detected in syncytia cells.

FIG. 3 is a representation of labelled proteins which were precipitatedby both cat anti-FPV serum and mouse anti-rabies antibody from³⁵S-methionine-labelled RCNV/FPV infected cell lysates, in animmunoprecipitation assay for protein expression.

FIG. 4 is a graph showing viral neutralization titers against FPV incats vaccinated (days 0, 26 and 43) with the multivalent recombinantRCNV vaccine (▪), and unvaccinated controls (□) following FPV challenge.

FIG. 5 is a graph showing total white blood cell counts of allvaccinated cats (▪) and control cats (□) following FPV challenge.

FIG. 6 is a diagram of the cloning of feline calicivirus (FCV) capsidprotein gene and construction of a raccoon poxvirus hemagglutinin gene(HA) insertion vector carrying the FCV capsid protein gene.

FIG. 6A is a schematic showing enzymatic amplification of the FCV capsidprotein gene, and subcloning the amplified FCV fragment into a vacciniainsertion vector.

FIG. 6B is a schematic showing construction of a fragment containing theFCV capsid gene operably linked to a promoter, and cloning the fragmentinto plasmid pGEM-3Z in forming a raccoon poxvirus HA insertion vectortermed pGEM/HA/FCV.

FIG. 7 shows immunofluorescent antibody staining of cells infected by arecombinant raccoon poxvirus containing the FCV capsid protein gene.

FIG. 7A shows positive plaques in a heavily infected well at lowermagnification (50×).

FIG. 7B shows positive plaques at higher magnification (500×).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for constructing recombinantRCNV which have incorporated into the viral thymidine kinase gene regionand/or hemagglutinin gene region multiple exogenous genes encodingantigens of various feline pathogens. Also provided are vaccinecompositions comprising the recombinant RCNV, and a method of using thevaccine compositions to elicit a protective immune response in immunizedfelines.

Definitions

“Expression cassette” is a term used herein for the purposes of thespecification and claims to refer to a recombinant nucleic acid moleculecontaining multiple genes, each encoding an antigen of a felinepathogen, which are operably linked to one or more promoters such thatwhen the expression cassette is inserted into RCNV, and upon subsequentinfection of feline cells with the recombinant RCNV, the antigensencoded by their respective gene are produced by the infected felinecells. “Feline pathogen” is a term used herein for the purposes of thespecification and claims to refer to one or more microorganisms whichare natural pathogens of cats, and include rabies virus, felinepanleukopenia virus (FPV), feline Chlamydia, feline immunodeficiencyvirus (FIV), feline leukemia virus (FeLV), feline infectious peritonitisvirus (FIPV), calicivirus, and feline herpesvirus (FHV).

The method of the present invention first comprises ligating anexpression cassette into the thymidine kinase (TK) gene contained intoan insertion vector such as a vaccinia virus (VV) expression vector.Because of the sequence homology between VV TK gene and the RCNV TKgene, the expression cassette within the VV expression vector, andflanking VV TK gene sequence, is then recombined into the TK gene ofraccoon poxvirus (RCNV). Alternatively, an expression cassette can beligated into an insertion vector which has HA sequences flanking theinsertion which are sufficiently homologous to promote recombination ofthe expression cassette into the HA gene of raccoon poxvirus. In eitherinstance, the resultant recombinant virus can then be used as a vaccinecomposition in a method for immunizing felines against challenge bythose feline pathogens, antigens of which are encoded by the expressioncassette in the recombinant virus. For purposes of illustration anddescription, but not limitation, in one embodiment of the presentinvention the FPV VP2 gene, and the rabies virus G protein gene wereeach separately and operably linked to a VV late promoter in forming anexpression cassette which was then recombined into raccoon poxvirus.However, the methods of the present invention provide a way to insertother exogenous genes into the raccoon poxvirus for expression uponinfection into a susceptible host cell. Thus, the present inventionincludes compositions comprising a combination of two or more genesencoding antigens found in one or more species/strains of felinepathogens, as will be illustrated in the following embodiments:

EXAMPLE 1 Construction of a Recombinant Insertion Plasmid

The essential features of an insertion plasmid that is useful in themethod of the present invention include the following features.

(a) The plasmid sequences flanking the insertion site into which are tobe inserted multiple genes, contain sequences which have sufficienthomology with sequences present in the raccoon poxvirus genome tomediate recombination. For example, a plasmid comprised of vacciniavirus sequences is used, wherein the site for insertion of multiplegenes (inserted as an expression cassette) is flanked by vaccinia virusthymidine kinase gene sequences. The nucleotide sequence of the TK geneof VV is not identical to the nucleotide sequence of the TK gene ofraccoon poxvirus; however, there is a sufficient degree of identity(“homology”) to promote hybridization of the TK gene sequence of VV tothe TK gene sequence of raccoon poxvirus and subsequent recombination.Thus, multiple genes flanked by TK gene sequence of VV are subsequentlyrecombined into the TK gene of raccoon poxvirus. Alternatively, suchflanking sequences can be part of the insert to be inserted into theplasmid.

(b) The flanking sequences must be homologous to a region of the raccoonpoxvirus (into which the multiple genes are recombined) that isnonessential for the growth and propagation of the raccoon poxvirus. Forexample, it was found, as illustrated in Examples 2, 6 and 8-10, thatboth the TK gene and the hemagglutinin (HA) gene of the raccoon poxvirusgenome can be used for insertion of exogenous genes by recombination.Insertion of exogenous genes into both or either of the TK gene or HAgene by recombination results in recombinant raccoon poxvirus capable ofinfection and replication, and can be used for expression of therecombined exogenous genes in host cells infected with the recombinantvirus.

Desirable features of an insertion vector that is useful in the methodof the present invention include the following features.

(a) Although it is possible that exogenous genes recombined into theraccoon poxvirus genome may be expressed without first operably linkingthe genes with one or more control element elements for expression (suchas a promoter) prior to recombination, operably linking the controlelement(s) to the multiple genes (thereby forming an expressioncassette) before using the genes for insertion into the plasmidinsertion vector, will likely result in higher efficiency of expressionof the recombined genes. Alternatively, the sequences flanking theinsertion site of the plasmid insertion vector can be engineered tocontain control elements which are then operably linked to the multiplegenes upon insertion.

(b) Although one promoter may be used to drive the expression of twoexogenous genes to be recombined, use of two promoters in an insertionvector, each promoter operably linked to an individual exogenous gene,will provide higher efficiency of expression.

To illustrate this embodiment, first an recombinant insertion plasmidwas constructed by inserting a 2,304 base pair (bp) fragment containingthe FPV VP2 gene (nucleotides 1-1752 of SEQ ID NO:1), from infectiousFPV genomic DNA, into a VV insertion vector. The 2,304 base pair (bp)fragment was released from the infectious clone by digestion withrestriction enzyme HincII and SmaI. A VV insertion vector, pTKgptF3S(Baroudy et al., 1980, J. Biol. Chem. 255:4372-4380), was digested withHincII, treated with calf intestinal phosphatase, purified, and ligatedwith the FPV VP2 DNA. The ligation mixture was used to transformEscherichia coli DH5 alpha. The transformed E. coli cells were plated onLauria broth (LB) agar plates with 50 μg/ml ampicillin and incubatedovernight at 37° C. The colonies grown on the agar plates were screenedby in situ colony hybridization using a ³²P-labeled FPV VP2 probe. Thecorrect orientation and the proper open reading frame were furtherconfirmed by restriction enzyme mapping and DNA sequencing. A plasmid,termed F3S/FPV, contained the VP2 gene in the correct orientation inrelation to the operably linked VV P₁₁ promoter.

A DNA fragment containing the rabies-G gene (SEQ ID NO:2) operablylinked to a VV P₁₁ promoter (SEQ ID NO:3) was purified from a RCNVrabies-G recombinant virus described previously (Ngichabe, C. K., 1992,Ph.D. Thesis, Cornell University, Ithaca, N.Y.). The RCNV rabies-Grecombinant virus was grown in CV-1 cell monolayers. When the cytopathiceffect (CPE) reached about 80% of the cell monolayer, the cells werescraped off, washed in PBS with 100 μM MgSO₄ (PBS-M) and resuspended in1,200 μl of PBS-M. The virus was released from the infected cells bydenaturation solution (0.5% Triton, 45 mM mercaptoethanol and 20 mMEDTA). The cell lysate was centrifuged at 300×g to separate thechromosomal DNA and debris, then the viruses were pelleted from thesupernatant by centrifugation at top speed of the microcentrifuge for 10minutes, and suspended in 100 μl of Tris-EDTA buffer (TE), pH 7.5. Aftertreatment with 150 μg/ml proteinase K, 200 mM NaCl, and 45 mMmercaptoethanol. The mixture was mixed gently and incubated for 2 hoursin a 50° C. waterbath. After extraction twice with equal volume of Trisbuffered phenol:chloroform:isoamyl alcohol and four times with watersaturated ether and ethanol precipitation. The DNA was pelleted bycentrifugation at top speed at 4° C. in a micro-centrifuge, washed in70% cold alcohol, resuspended in 100 μl of water and then stored at −70°C. for use.

A DNA fragment containing the gene encoding rabies-G was isolated, andcloned into F3S/FPV, as follows (See also FIG. 1). A 1.75 Kb fragmentcontaining the rabies-G gene operably linked to a P₁₁ late promoter wasamplified by polymerase chain reaction (PCR) from purified RCNV rabies-Grecombinant virus DNA using a forward primer (SEQ ID NO:4) and a reverseprimer (SEQ ID NO:5). After separation of the amplified DNA on 1%agarose gel, the amplified DNA was digested by BamHI and KpnI which weredesigned on both ends of the PCR primers. The reverse primer had anartificial restriction enzyme digestion site of BamHI which was aimed atthe tail BamHI site of the FPV VP2 gene (FIG.1). The forward primercarried an artificial restriction enzyme digestion site of KpnI whichwas aimed at the KpnI site in the gpt fragment of the F3S/FPV plasmid(FIG. 1). The DNA bands were extracted, reseparated on the 1% agarosegel and recovered by a DNA purification kit. The DNA fragments,comprising linearized F3S/FPV and the amplified DNA which had beenrestricted, were ligated together and the ligation mixture was used totransform E. coli DH5α. The transformed E. coli cells were plated on theLB agar plate containing 50 μg/ml ampicillin, and incubated at 37° C.overnight. The amp-resistant selected bacterial colonies were screenedby colony hybridization using both FPV VP2 and rabies G genes as probes(SEQ ID NO:1 and SEQ ID NO:2, respectively). The colonies positive byhybridization were then selected and cultured in the LB medium forpreparation of plasmid DNA. A plasmid, termed F3S/FPV/RAB, was formedwhich contained rabies-G gene operably linked to a P₁₁ and the FPV VP2gene operably linked to a VV P₁₁ promoter (FIG. 1). The purified plasmidDNA was further confirmed by DNA sequencing. Three primers were used tosequence both FPV VP2 and rabies G genes (SEQ ID NOs: 4, 5, and 6). Aprimer (SEQ ID NO:6) was used to sequence the promoter, the junction ofpromoter, and VP2 coding sequence. The two PCR primers (SEQ ID NO:4 andSEQ ID NO:5) were used to sequence the inserted PCR fragment. All of thesequenced DNAs were compared with the original published sequences. Notethat in F3S/FPV/RAB, the respective exogenous genes, operably linked totheir respective promoter, are in opposite orientation (in the directionof transcription) relative to each other. Each inserted gene had its ownpromoter, start codon and stop codon for authentic protein expression.Thus, both inserted genes were not mutated in this multivalent plasmid.

EXAMPLE 2 Construction of a Recombinant Raccoon Poxvirus

The essential features of a potential viral vaccine vector for use inthe immunization of felines according to the present invention includethe following features.

(a) The viral vector need be safe and suitable as a vaccine in cats. Forexample, the viral vector must infect and replicate in cats withoutcausing significant pathology in immunized cats. Also, the viral vectormust be capable of expressing genes recombined into it, upon infectingfeline cells.

(b) The viral vector must be able to tolerate large foreign insertions(multiple genes inserted by recombination) into either or both of thethymidine kinase gene and the hemagglutinin gene without significantlyand negatively affecting the ability of the recombinant virus to infectand replicate in susceptible host cells.

Raccoon poxvirus (RCNV) was found to be safe and suitable as a viralmonovalent vaccine vector useful for immunizing cats (Ngichabe, C. K.,1992, supra). However, it was not known at the time of the inventionwhether raccoon poxvirus could tolerate large foreign insertions, asrequired when recombining multiple genes/expression cassettes (versus asingle gene), into the thymidine kinase gene or its hemagglutinin geneof the raccoon poxvirus genome without the virus losing its ability toinfect and replicate in susceptible host cells. Further, it was notknown at the time of the invention whether there were any constraintsrelative to the orientation of insertion of multiple genes with respectto the direction of transcription. Extrapolating the results of studieswith VV vectors, relative to the capacity of the viral genome totolerate insertions and relative to orientation, to RCNV would bepresumptuous because the DNA sequences between RCNV and VV aresignificantly different.

The multivalent RCNV-based recombinant FPV and rabies virus wasgenerated by homologous recombination of the flanked vaccinia virus TKgene sequence connected to both ends of the two insertions (VP2 andrabies-G) with the TK fragment of RCNV. Purified plasmid DNA containingboth FPV VP₁₁ and rabies-G genes, with each gene operably linked to aP₁₁ promoter, was transfected by calcium phosphate precipitation intoCV-1 cell monolayers which were also infected with raccoon poxvirus.When the plaques reached about 60% confluence, the transfection mixturewas harvested and freezethawed three times. The recombinant RCNV viruswas selected by plaque purification in the presence of 50 μg/ml5-bromo-2′deoxyuridine (BUDR), and dot blot hybridization using³²P-labelled FPV VP2 and rabies-G gene probes (SEQ ID NO:1 and SEQ IDNO:2, respectively). The positive plaque was further plaque purifiedthree times in rat-2 TK cells with the presence of BUDR and then grownin CV-1 cells without BUDR. The cells were harvested 1 to 2 days laterwhen maximum CPE was observed. The cell suspension was frozen and thawedthree times and sonicated before use as vaccine. If desired, therecombinant RCNV can be pelleted by gradient centrifugation,resuspended, and titrated on Rat-2 cell monolayers, to determine thepfus.

EXAMPLE 3 Molecular Analysis of the Multivalent Recombinant RaccoonPoxvirus

In this embodiment, the multivalent recombinant raccoon poxvirus wasanalyzed for the presence of the VP2 gene and rabies-G gene. Themultivalent recombinant raccoon poxvirus was purified byplaque-purification and selected by dot-blot hybridization using³²P-labelled VP2 and rabies-G gene probes (SEQ ID NO:1 and SEQ ID NO:2,respectively), as described in Example 2. Further, using DNA isolatedfrom the purified multivalent recombinant raccoon poxvirus, an internalsequence of each of the FPV VP2 and rabies-G genes were enzymaticallyamplified. Two bands were observed when the enzymatically amplified DNAwas run on a 1% agarose gel. The bands were the expected sizes of therabies-G gene internal sequence, and the FPV VP2 internal sequence. Thegel was also probed with both the radiolabelled VP2 and rabies G geneprobes after Southern transfer; and the results verify the presence ofthe VP2 and rabies-G genes in the multivalent recombinant raccoonpoxvirus.

EXAMPLE 4 Expression of VP2 and Rabies-G Proteins in the Cells Infectedwith Multivalent Recombinant Raccoon Poxvirus

In this embodiment, the multivalent recombinant raccoon poxvirus wasanalyzed for expression of the recombined multiple genes.

4.1 Indirect Immunofluorescent Antibody Assay (IFA)

An IFA was performed to detect expression of VP2 and rabies-G by cellsinfected with the multivalent recombinant raccoon poxvirus. Briefly, theCV-1 or CrFK (Crandell feline kidney) cell monolayers, grown in 8-wellchamber slides, were infected with the multivalent recombinant virus atone multiplicity of infection. After fixation in cold acetone for 10minutes, the cell monolayer was washed in PBS and immersed with 1:10diluted normal cat serum (from specific-pathogen-free cats; “SPF” cats)for 10 minutes. The slides were then incubated with cat anti-FPVpolyclonal antibody diluted 1:200 or mouse anti-rabies-G monoclonalantibody for one hour. The slides were thoroughly washed in PBS andincubated with fluorescein-labelled rabbit anti-mouse antibody for 30minutes. After washing in PBS, the slides were sealed by glycerol andexamined by immuno-fluorescence microscopy.

Immunofluorescence was detected when either cat anti-FPV serum or mouseanti-rabies G monoclonal antibody was reacted with cells infected withmultivalent recombinant virus. infected cells or plaques. FIG. 2 showsthe IFA-positive plaques/cells infected with the multivalent recombinantraccoon poxvirus. Bright green fluorescence was mostly concentrated onthe cytoplasm of the infected cells. Also, IFA detected positiveparticles in the center of some positive plaques. No difference instrength or patterns of fluorescence was observed when IFA was performedwith FPV polyclonal antibody versus with rabies monoclonal antibody.

4.2 Immunoprecipitation Assay

An immunoprecipitation assay was performed to detect expression of VP2and rabies-G by cells infected with the multivalent recombinant raccoonpoxvirus. ³⁵S-methionine-labelled cell lysate, from CV-1 cells infectedwith the multivalent recombinant raccoon poxvirus, wasimmunoprecipitated with cat anti-FPV antiserum and mouse anti-rabies-Gmonoclonal antibody. Briefly, the CV-1 cell monolayers were infectedwith the multi valent recombinant raccoon poxvirus, and then werepulse-labelled with ³⁵S-methionine for 12 hours. The infected cells werelysed in lysis buffer, and all solubilized cellular proteins wereseparated from the cell debris by centrifugation. The clarified proteinsuspension was precipitated with anti-FPV antibody or anti-rabies-Gmonoclonal antibody. The precipitated proteins were loaded onto an 8% to25% gradient polyacrylamide gel and electrophoresed. The mini-gel wasdirectly fixed, enhanced, and finally neutralized before exposure toX-ray film.

Precipitated proteins, precipitated by both cat anti-FPV serum and mouseanti-rabies antibody from the ³⁵S-methionine-labelled RCNV/FPV infectedcell lysate, are indicated in FIG. 3. The expression of three proteinswas observed by the immunoprecipitation assay: FPV VP2 protein of 67kilodalton (Kd) (FIG. 3, lane 1); and rabies-G1 of 64 Kd, and rabies-G2of 62 Kd (lane 2). The two rabies bands were very clear if the exposuretime was shorter. Lane 4, containing a control cell lysate infected withwild type RCNV, did not show similar bands in the immunoprecipitationassay.

EXAMPLE 5 Vaccine Studies with Multivalent Recombinant Raccoon Poxvirus

In this embodiment, the multivalent recombinant raccoon poxvirus wasused to immunize cats to induce an immune response against the felinepathogen antigens expressed by cells infected the multivalentrecombinant raccoon poxvirus (RCNV/FPV/RAB-G).

5.1 Preparation of vaccine stock

The vaccine stock for the RCNV/FPV/RAB-G construct was prepared using apreviously described method for preparing raccoon poxvirus recombinantsfor use in vaccines (Esposito et al., 1988, Virology 165:313-316). CrFKcell monolayers, grown in 150 cm² tissue culture flasks, were infectedwith plaque-purified RCNV/FPV/RAB-G at multiple of infectivity 32 0.5.The multivalent recombinant virus was allowed to adsorb at 37° C. for 2hours. Supplemented tissue culture medium was added, and the cultureswere incubated 24 hours at 37° C. When the confluent CPE was reached,the cells were scraped, concentrated by polyethylene glycol (PEG 8000)and pelleted at 16,000×g for 30 minutes. The pellet was suspended in 1.0ml of Tris-C1 EDTA (TE), pH 7.6, for every 100 ml of starting culturefluid. Sodium diatrizoate composite gradients were used for layering thesuspended mixture. The gradient contained 1 ml of 50% diatrizoate, 25 mlof 25-50% diatrizoate, and 3 ml of Dextran-10. After centrifugation at125,000×g for 16 hours at 4° C., the viral band was collected, dilutedfour-fold in TE buffer, and centrifuged through a 30% sucrose cushion.The viral pellet was then resuspended, sonicated, titrated on Rat-2cells and diluted in 50% v/v in sterile glycerol and TE buffer. Thevaccine mixture was finally stored at −20° C. for later use.

5.2 Design of Vaccination and Challenge Studies

The animals used in the vaccination and challenge studies were five- to6-month-old specific-pathogen-free cats (SPF cat colony) that were keptin regulatory-approved biohazard isolation units. Group A consisted of 9cats for vaccination. Group B had 2 cats that were unvaccinatedcontrols. Group A was vaccinated subcutaneously three times on days 0,26, and 34 with recombinant RCNV carrying both FPV VP2 and rabies Ggenes (RCNV/FPV/RAB-G). Each cat received a dose of 1×10⁷ PFU virus eachtime. The control cats received phosphate buffered saline (PBS) at thesame time as group A was vaccinated. Sixteen days following the thirdvaccination, all cats were challenged orally with 10^(4.5) TCID₅₀ of FPVchallenge virus (National Veterinary Service, Ames, Iowa.) strictlyfollowing the USDA challenge procedure.

5.3 Vaccination and Challenge Results

5.3.1 Virus Neutralization Antibody Titer

All cats in the vaccination and challenge studies were checked daily forclinical signs. Blood was collected for total white blood cell counts atdays 0, 3, 5, 7 and 10 after challenge, and the sera were collected ondays 26, 43, 50 and 57 after vaccination. All sera were aliquotted intotwo vials for both FPV antibody and rabies antibody tests. The viralneutralization antibody assay for antibodies against rabies virus wasdone by the Rabies Virus Laboratory, New York State Department ofHealth, Albany, N.Y. The viral neutralization test for rabies virus wasdone by the rapid immunofluorescent focus inhibition test. The rabiesneutralization antibody titers were interpreted as international unit(IU) per ml (1 IU=1:32 virus neutralization titer).

The serum from each cat was also assayed by viral neutralizationantibody assays for anti-FPV antibody titer. Briefly, CrFK cells wereseeded on 8-chamber slides at 1×10⁵ cells/ml, and incubated at 37° C.for one hour. Two-fold serial dilutions of all collected serum sampleswere mixed with an equal volume of 32-100 TCID₅₀/0.1 ml of FPV, and wereincubated at 37° C. for another hour. The mixtures were then added toindividual chambers seeded with CrFK cells and incubated at 37° C. in a5% CO₂ atmosphere for 3 to 4 days. The infected cell monolayers werefixed with methyl alcohol, stained with May Grüwald-Giemsa stain andexamined under light microscope for FPV intranuclear inclusions. Thehighest dilution of serum at which no detectable FPV inclusion bodieswere identified was interpreted as the virus neutralization titer.

For the FPV virus neutralization assay, the eight cats vaccinated withthe multivalent recombinant raccoon poxvirus (RCNV/FPV/RAB-G) achieved amean virus neutralization titer of 1:1,000 at day 27 (FIG. 4). The virusneutralization titer reached 1:7,000 at day 43, and over 1:10,000 by day50 (FIG. 4). All three vaccinations boosted the FPV virus neutralizationtiter, which was not affected by the FPV challenge in vaccinated cats.However, the unvaccinated cats (Group B) obtained virus neutralizationtiter only after day 50 when the FPV challenge was given. The virusneutralization titer in Group B cats, following challenge with FPV, wasas high as 1:5,000 at day 60 (FIG. 4).

For the rabies virus neutralization assay (Table 1), the virusneutralization titer was as high as 4 to 16 international unit (IU) atday 43; and up to 8 to 16 IU at day 50 after vaccination. The virusneutralization titers of two vaccinated cats were ≧8 international units(IU) at day 26; and the titers of 4 vaccinated cats rose to a ≧16 IU byday 43. All of the vaccinated cats obtained a virus neutralization titerover 8 IU at day 50, and four out of 8 vaccinated cats obtained a virusneutralization (VN) titer over 16 IU by day 57 after vaccination.However, the control unvaccinated cats were negative throughout theexperiment (Table 1).

TABLE 1 Date RABIES VN TITERS (IU) Cat # 0 26 43 50 57 Unvaccinated A981<0.063 <0.063 <0.063 <0.063 <0.063 cats A932 <0.125 <0.125 <0.125 <0.125<0.125 Vaccinated A983 <0.063 1 4 >=8 >=8 Cats A954 <0.063 2 >=8 >=8 >=8A962 <0.063 2 4 >=8 >=8 A931 <0.063 >=8 >=8 >=8 >=8 A964 <0.1254 >=16 >=16 >=16 A982 <0.125 1 >=16 >=16 >=16 A955 <0.1251 >=16 >=16 >=16 A963 <0.125 8 >=16 >=16 >=16 (1 IU = 1:32 VN titer)

5.3.2 Leukocyte Counts

As described above, blood was collected for total white blood cellcounts at days 0, 3, 5, 7 and 10 after challenge. The total white bloodcells and differential counts were determined using an automatedhematology analyzer. The clinically critical indication of the FPVinfection is the change in white blood cell (WBC) counts during thefirst 10 days after FPV infection. As shown in FIG. 5, total WBC countsof all vaccinated cats were stable in the clinically no=al rangethroughout the experiment (▪). The mean WBC counts of the unvaccinatedcats were typical of FPV infection. The mean counts were as low as 2.3thousand/microliter at day 5 after the FPV challenge (□) and the catswere fully recovered at day 20 when the experiment was ended. Thiscritical indicator of an immune response to FPV demonstrated strongprotection of vaccinated cats from subsequent FPV challenge.

Taken together, the vaccination studies indicate that cats vaccinatedwith the multivalent recombinant raccoon poxvirus were fully protectedfrom FPV challenge, and provoked strong humoral immune responses whichmay be sufficient to protect the cats from rabies infection. Due to theregulatory restriction of rabies challenge, the efficiency of rabiesprotection was based on the serum virus neutralization antibody titersfollowing vaccination. However, previous studies have showed that avirus neutralization titer of 0.5 IU is sufficient for protectionagainst rabies virus challenge (Barth et al., 1988, Vaccine 6:369-377;Ngichabe, 1992, supra). Thus, the virus neutralization titers of catsvaccinated with the multivalent recombinant raccoon poxvirus vaccinewere over 32 fold higher than the recommended satisfactory titers. Basedon the data, it is believed cats vaccinated with such a multivalentrecombinant raccoon poxvirus would be protected from subsequent rabiesvirus challenge.

EXAMPLE 6

6.1 Feline Calicivirus as a Feline Pathogen Antigen in a MultivalentRecombinant Raccoon Poxvirus Vaccine

Feline calicivirus (FCV) is an important pathogen of cats causingserious feline diseases including acute oral ulceration, mild upperrespiratory diseases and severe lower respiratory diseases, and febrilelameness syndrome. In infected cats, FCV is shed in ocular, nasal, andpharyngeal secretions. Cats that have recovered from FCV infection mayalso become persistent shedders of the virus. The capsid protein of FCVin FCV-infected cells is a 76 Kd protein. Neutralizing epitopes exist onthe capsid protein. The nucleotide sequence of the FCV capsid proteinwas reported recently (Seal et al., 1993, J. Gen. Virol. 74:2519-2524;Seal et al., 1995, Virus Genes 9:183-187). A cDNA clone of the capsidprotein gene (SEQ ID NO:7), from highly virulent strain FCV 255, wasused in the construction of a multivalent vaccine as follows.

Using the methods of the present invention, as illustrated in Examples1-3, an expression cassette can be constructed to include anycombination of two or more genes, wherein the genes encode the felinecalicivirus (FCV) capsid protein (SEQ ID NO:7), FPV VP2 (SEQ ID NO:1),or rabies-G (SEQ ID NO:2). The expression cassette may then be insertedinto an insertion vector, and subsequently recombined into the TK geneof raccoon poxvirus, thereby forming a multivalent recombinant raccoonpoxvirus.

Alternatively, genes encoding two or more feline pathogen antigens canbe recombined into the hemagglutinin (HA) gene of raccoon poxvirus informing a multivalent recombinant raccoon poxvirus. In anotherembodiment, using the methods according to the present invention, boththe HA gene and the TK gene can be used as sites into which at least onegene encoding a feline pathogen antigen is recombined into each site(recited in the claims as “a combination thereof”). The vaccinia virusHA gene has been used for construction of vaccinia HA insertion vectors(Shida, 1989, Subcell. Biochem. 15:405-440; Shida et al., 1983, Cell33:423-434). The HA sequence is not essential for virus reproduction orinfectivity in cell culture, but it affects the way that the virus isdisseminated. The HA glycoprotein, synthesized by rough endoplasmicreticulum in the plasma membrane, gives both hemagglutination andhemadsorption. Thus, using hemagglutination and hemadsorption assays,mutations of the HA gene allow screening and selection of the HAphenotype.

Analysis of the HA genes of both RCNV and vaccinia virus showed homologyof 69% in DNA sequences and 53% in amino acids of the HA between RCNVand VV (Cavallaro et al., 1992, Virology 190:434-439). Thus, the low HAhomology between the two viruses excludes the possibility of, and wouldteach against one skilled in the art to attempt, using vaccinia HAinsertion vectors for homologous recombination into the RCNV HA gene.

6.2 Construction of a Recombinant HA Insertion Plasmid

The essential features of an HA insertion plasmid that is useful in themethod of the present invention include the following features.

(a) The plasmid sequences flanking the insertion site into which are tobe inserted multiple genes, contain sequences which have sufficienthomology with sequences present in the raccoon poxvirus genome tomediate recombination. Thus, to construct such a HA insertion vector,raccoon poxvirus HA sequence was used as the flanking sequences toposition a vaccinia virus promoter and the insert containing one or moregenes that is expected to be expressed. Although the HA flankingsequences can be synthesized to vary slightly from the HA sequence foundin RCNV, there must be a sufficient degree of identity (“homology”) topromote hybridization of the HA flanking sequence to the HA genesequence of raccoon poxvirus and subsequent recombination.Alternatively, such flanking sequences can be part of the insert to beinserted into the plasmid.

(b) The flanking sequences must be homologous to a region of the raccoonpoxvirus (into which the multiple genes are recombined) that isnonessential for the growth and propagation of the raccoon poxvirus. Asillustrated in this Example, the hemagglutinin (HA) gene of the raccoonpoxvirus genome can be used for insertion of exogenous genes byrecombination. Insertion of exogenous genes into the HA gene byrecombination results in recombinant raccoon poxvirus capable ofinfection and replication, and can be used for expression of therecombined exogenous genes in host cells infected with the recombinantvirus.

Desirable features of an insertion plasmid that is useful in the methodof the present invention include the following features.

(a) Although it is possible that exogenous genes recombined into theraccoon poxvirus genome may be expressed without first operably linkingthe genes with one or more control element elements for expression (suchas a promoter) prior to recombination, operably linking the controlelement(s) to the multiple genes (thereby forming an expressioncassette) before using the genes for insertion into the plasmidinsertion vector, will likely result in higher efficiency of expressionof the recombined genes. Alternatively, the sequences flanking theinsertion site of the plasmid insertion vector can be engineered tocontain the control elements which are then operably linked to themultiple genes upon insertion.

(b) Although one promoter may be used to drive the expression of twoexogenous genes to be recombined, use of two promoters in an insertionvector, each promoter operably linked to an individual exogenous gene,will provide higher efficiency of expression.

Thus, in this embodiment, RCNV HA sequence was used as flankingsequences to position a promoter operably linked to the foreign geneinsert that is to be expressed. To illustrate this embodiment, a 2381bps cDNA fragment amplified from the pSV.SPORT1/FCV cDNA clone (FIG. 5)was flanked with an XhoI site at the 5′-start codon end and KpnI at the3′ end. A vaccinia TK insertion plasmid, pMJ601, was digested with SalIand KpnI. The FCV fragment, containing the capsid protein gene, wasligated into pMJ601 to form a plasmid, termed pMJ601/FCV. By suchinsertion, the FCV capsid protein gene was operably linked to a vaccinialate promoter. The plasmid DNA was sequenced to confirm the accuracy ofthe cloned fragment, and the open reading frame orientation.

A RCNV HA fragment of 1,262 base pairs (bp) was divided into two partsfrom which a HA left arm (HAL) and a right arm (HAR) were prepared byenzymatic amplification using the polymerase chain reaction (PCR). TheHA left arm of 582 bps (SEQ ID NO:8) and the HAR of 447 bps (SEQ IDNO:9) were amplified using primers P1, P2, P3 and P4 (FIG. 6). Themiddle portion of the HA gene (between HAL and HAR) was deleted afterforming the two end fragments. The amplified products of HAL, HAR, andof the capsid protein gene operably linked to a promoter (P/FCVCP), werejoined and amplified by recombinant PCR. The full sequence, whichcontained HAL-P/FCVCP-HAR of 3,353 bp was prepared from two recombinantfragments. Conditions typically used for enzymatic amplification,include a denaturation step at 95° C. for 1 minute, an annealing step at60° C. for 1 minute, an extension at 72° C. for 3 minutes, an extraextension at 72° C. for 7 minutes, and a soaking step at 4° C. foranalysis. For FCV capsid protein gene amplification, an annealingtemperature at 68° C. was applied and other temperatures were the sameas above. Enzymatically amplified fragments of HAL and P/FCVCP werejoined by recombinant PCR using the 20 bps overhang sequence of aprimer, P3 (SEQ ID NO:10). About 10 to 50 ng of purified DNA fragmentsof HAL and P/FCVCP were mixed with a thermostable DNA polymerase withoutprimers. The first three cycles of PCR were designed for ligation of thetwo fragments in the presence of the thermostable DNA polymerase. Theprotocol of the thermocycle steps was as follows: denature at 94° C. for1 minute, anneal at 40° C. for 2 minutes at a ramp rate of 6 seconds/perdegree and extension at 72° C. for 3 minutes at ramp rate of 2seconds/per degree. Primers P1 (SEQ ID NO:11) and F2 (SEQ ID NO:12) wereadded to the reaction tube and a regular PCR was performed for 30 cycleswith denaturation at 94° C. for 1 minute, annealing at 55° C. for 1minute and extension at 72° C. for 2 minutes (See also, FIG. 6A). Afinal incubation at 72° C. for 7 minutes was used for the extension ofall amplified fragments. Using the same protocol and conditions,amplified fragments of P/FCVCP and HAR were ligated by primers P5 (SEQID NO:13) and P2 (SEQ ID NO:14) (See also, FIG. 6B). All of theamplified products were purified from 1% agarose gel by a DNApurification kit.

The low annealing temperature (40° C.) and ramp temperature during theturnover of each thermocycle step greatly facilitated the process ofrecombination. After two to three cycles without primer, the ligatedfragment was amplified by PCR. The resultant recombinant product,HAL-P/FCVCP-HAR, was ultimately subcloned into plasmid pGEM−3Z to form anovel RCNV HA insertion plasmid carrying FCV, termed pGEM/FCV. The totallength of the plasmid was 6175 bps which included 3353 bps ofHAL-P/FCVCP-HAR and 2726 bps of PGEM (FIG. 6B).

6.3 Construction of a Recombinant Raccoon Poxvirus

Homologous recombination between the RCNV HA sequences of pGEM/FCV andbetween the HA sequences of wild type RCNV was performed with proceduresoutlined in Example 2, and as described previously (See for example,Mackett et al., J. Virol. 49:857-684, herein incorporated by reference).Briefly, the purified HA insertion vector, pGEM/FCV, was precipitatedonto a CV-1 cell monolayer infected with wild type RCNV by calciumphosphate transfection. After 48 hours of the transfection, the cellmonolayer was frozen and thawed three times. The virus mixture wasplated at different dilutions onto the CV-1 monolayer and grown in the8-well chamber slide. When CPE appeared, the cells were fixed andstained with cat anti-FCV serum.

6.4 Expression of FCV Capsid Protein in Cells Infected with aRecombinant Raccoon Poxyirus

Indirect immunofluorescent antibody assays were performed to detectFCV-specific fluorescence in the cytoplasm and plasma membrane ininfected cells and recombinant raccoon poxvirus-formed plaques in cellculture. CV-1 cell monolayers were infected with the recombinant RCNV orwild type RCNV and incubated for 18 to 24 hours at 37° C. The infectedcell monolayers were fixed in cold acetone, incubated first with 1:10diluted normal cat serum for 10 minutes, and then with 1:100 diluted catanti-FCV antiserum for one hour at room temperature. The monolayer wasfinally stained in 1:100 diluted fluorescein labelled rabbit anti-catIgG for 30 minutes and examined by fluorescent microscopy. Theimmunofluorescent antibody assay showed that positive viral plaqueand/or positive infected cells were detectable by specific FCV antibody.FIG. 7A shows two separate positive plaques in a heavily infected wellat lower magnification. FIG. 7B shows recombinant virus-induced plaquesat higher magnification. The cytoplasm of the infected cells and thesyncytia cells were heavily stained with intensive fluorescence. Eventhough the fluorescent particles were more likely concentrated aroundthe nucleus, the positive particles were distributed all over thecytoplasm and plasma membrane. All of the wild type RCNV-infected cellswere negative.

6.4 Vaccines Comprising Recombinant Raccoon Poxvirus Containing FCVCapsid Protein Gene

Using the methods according to Example 5, vaccine stocks of recombinantraccoon poxvirus containing FCV capsid protein gene can be prepared, andadministered in a vaccination process to felines.

EXAMPLE 7 Alternatives in Feline Pathogen Antigen Expression

The selection of a promoter may depend on the feline pathogen antigen tobe expressed. Promoters vary in strength, i.e. ability to facilitatetranscription. Generally, for the purpose of expressing a cloned gene,it is desirable to use a strong promoter in order to obtain a high levelof transcription of the gene and expression into gene product. Forexample, viral promoters known in the art, which may be used in amultivalent recombinant raccoon poxvirus, from which a high level oftranscription has been observed in infected mammalian cells include theSV40 early promoter, CMV promoters, various vaccinia promoters,adenovirus major late promoter, and the like, may be used to providetranscription of the inserted DNA sequence encoding feline pathogenantigens.

To improve the efficiency of expression of feline pathogen antigens, itis preferable that each gene encoding an antigen is operably linked to apromoter. However, for some promoters, such as the vaccinia virus P₁₁promoter, apparently two genes with the same kind of promoter can not beoriented in the same direction in the recombinant raccoon poxvirus.Thus, a gene to be expressed may have to be inserted in a transcriptionorientation which is opposite to the transcription orientation of anadjacent gene to be expressed.

Other control elements for efficient gene transcription or messagetranslation include enhancers, and regulatory signals. Enhancersequences are DNA elements that appear to increase transcriptionalefficiency in a manner relatively independent of their position andorientation with respect to a nearby gene. Thus, an enhancer may beplaced either upstream or downstream from the inserted DNA sequencesencoding feline pathogen antigens to increase transcriptionalefficiency.

Further, genetic engineering techniques can be used to reduce a gene toa gene fragment that encodes only a portion of the feline pathogenantigen; but a portion that acts as an immune response-inducing epitope.For example, from the sequences of the various genes feline pathogen, itcan be determined which restriction enzyme or combination of restrictionenzymes may be used to generate sequences encoding immunogenic peptides.Restriction enzyme selection may be done so as not to destroy theimmunopotency of the resultant peptide. Antigenic sites of a protein mayvary in size but can consist of from about 7 to about 14 amino acids.Thus, a protein the size of most feline pathogen antigens may containmany discrete antigenic sites; therefore, many partial gene sequencescould encode antigenic epitopes. Consequently, using the nucleotidesequence of the particular gene as a guide, restriction enzymecombinations may be used to generate DNA sequences, which when insertedinto a multivalent recombinant raccoon poxvirus, are capable ofdirecting the production of peptides comprising one or more antigenicepitopes.

Modification of the feline pathogen antigens or peptides, such as bydeletion and substitution of amino acids (and including extensions andadditions to amino acids) and in other ways, may be made so as to notsubstantially detract from the immunological properties of the antigenor peptide. In particular, the amino acid sequence of the antigen orpeptide, may be altered by replacing one or more amino acids withfunctionally equivalent amino acids resulting in an alteration which issilent in terms of an observed difference in the physicochemicalbehavior of the antigen or peptide. Functionally equivalent amino acidsare known in the art as amino acids which are related and/or havesimilar polarity or charge. Thus, an amino acid sequence which issubstantially that of the amino acid sequences depicted in the SequenceListing herein, refers to an amino acid sequence that containssubstitutions with functionally equivalent amino acids without changingthe primary biological function of antigen, or peptide.

Also, it will be appreciated by those skilled in the art, that becauseof third base degeneracy, almost every amino acid is represented by morethan one triplet codon in a coding nucleotide sequence. Thus, a geneencoding a feline pathogen antigen as disclosed herein, may be modifiedslightly in nucleotide sequence, and yet still encode its respectivegene product of the same amino acid sequence. Thus, insertion of suchmodified genes into a multivalent recombinant raccoon poxvirus is withinthe scope of the present invention.

EXAMPLE 8 Feline Infectious Peritonitis Virus as a Feline PathogenAntigen in a Multivalent Recombinant Raccoon Poxvirus Vaccine

Feline infectious peritonitis virus (FIPV) is a coronavirus which causesa highly fatal disease in infected cats. It has been shown that incoronavirus infections, antibodies may be developed against the membraneglycoprotein (M) and the nucleocapsid protein (N) which can inhibitvirus replication. The nucleotide sequences of the FIPV M (SEQ ID NO:17)and N (SEQ ID NO:18) genes, and their deduced amino acid sequences, havebeen described previously (Vennema et al., 1991, Virology 181:327-335;the disclosure of which is herein incorporated by reference).

Using the methods of the present invention, as illustrated in Examples1-3, and 7, an expression cassette can be constructed to include anycombination of two or more genes, wherein one of the genes encodeseither the FIPV M or N protein. Other exogenous genes that can beincluded in such an expression cassette include genes that encode FCVcapsid protein, FPV VP2, or rabies-G. The expression cassette may thenbe inserted into an insertion vector, and subsequently recombined intothe TK gene of raccoon poxvirus, thereby forming a multivalentrecombinant raccoon poxvirus.

Alternatively, using the methods of the present invention, asillustrated in Examples 1-4, 6, and 7, an expression cassette can beconstructed to include any combination of two or more genes, wherein oneof the genes encodes either the FIPV M or N protein. Other exogenousgenes that can be included in such an expression cassette include genesthat encode the FCV capsid protein, FPV VP2, or rabies-G. The expressioncassette may then be inserted into an insertion vector, and subsequentlyrecombined into the HA gene of raccoon poxvirus, thereby forming amultivalent recombinant raccoon poxvirus.

In another embodiment, using the methods according to the presentinvention, both the HA gene and the TK gene can be used as sites intowhich exogenous genes encoding feline pathogen antigens are recombinedinto raccoon poxvirus. Combinations of such exogenous genes includegenes which encode FIPV M, FIPV N, FCV capsid protein, FPV VP2, andrabies-G. With all of these embodiments, using the methods according toExample 5 and other methods known in the art, vaccine stocks ofrecombinant raccoon poxvirus containing exogenous genes encoding felinepathogen antigens can be prepared, and administered in a vaccinationprocess to felines.

EXAMPLE 9 Feline Immunodeficiency Virus as a Feline Pathogen Antigen ina Multivalent Recombinant Raccoon Poxvirus Vaccine

Feline immunodeficiency virus (FIV) is a retrovirus which causes apersistent generalized lymphadenopathy, recurrent fevers, anorexia, andweight loss in infected cats. Often chronic secondary infections arepresent which are caused by other feline pathogens. A comprehensivereview of FIV biology, infection, and immune responses thereto, has beenrecently published (Bendinelli et al., 1995, Clin. Microbiol. Reviews8:87-112; the disclosure of which is hereby incorporated by reference).In vaccine studies, cats receiving immunizations with either inactivatedwhole-infected cell or cell-free feline immunodeficiency virus (FIV)vaccines were protected against subsequent FIV challenge; whereinprotection appeared to correlate with antiviral envelope antibody titers(Yamamoto et al., 1993, J. Virol. 67:601-605). The gene encoding FIV Gagprotein (approximately FIV nucleotide sequence base 600 to base 2,000;Bendinelli et al., supra), or the gene the FIV Env protein(approximately FIV nucleotide sequence base 6,250 to base 8,850;Bendinelli et al., supra) may be used to induce neutralizing antibodieswhich may inhibit virus infection and/or replication.

Using the methods of the present invention, as illustrated in Examples1-4, and 7, an expression cassette can be constructed to include anycombination of two or more genes, wherein one of the genes encodeseither the FIV Gag or Env protein. Other exogenous genes that can beincluded in such an expression cassette include genes that encode FIPVM, FIPV N, FCV capsid protein, FPV VP2, or rabies-G. The expressioncassette may then be inserted into an insertion vector, and subsequentlyrecombined into the TK gene of raccoon poxvirus, thereby forming amultivalent recombinant raccoon poxvirus.

Alternatively, using the methods of the present invention, asillustrated in Examples 1-4, 6, and 7, an expression cassette can beconstructed to include any combination of two or more genes, wherein oneof the genes encodes either FIV Gag or FIV Env. Other exogenous genesthat can be included in such an expression cassette include genes thatencode FIPV M, FIPV N, FCV capsid protein, FPV VP2, or rabies-G. Theexpression cassette may then be inserted into an insertion vector, andsubsequently recombined into the HA gene of raccoon poxvirus, therebyforming a multivalent recombinant raccoon poxvirus.

In another embodiment, using the methods according to the presentinvention, both the HA gene and the TK gene can be used as sites intowhich exogenous genes encoding feline pathogen antigens are recombinedinto raccoon poxvirus. Combinations of such exogenous genes includegenes which encode FIV Gag, FIV Env, FIPV M, FIPV N, FCV capsid protein,FPV VP2, and rabies-G. With all of these embodiments, using the methodsaccording to Example 5 and other methods known in the art, vaccinestocks of recombinant raccoon poxvirus containing exogenous genesencoding feline pathogen antigens can be prepared, and administered in avaccination process to felines.

EXAMPLE 10 Feline Leukemia Virus as a Feline Pathogen Antigen in aMultivalent Recombinant Raccoon Poxvirus Vaccine

Feline leukemia virus (FeLV) is a oncornavirus which causes leukemia andrelated symptoms in infected cats. FeLV env gene expression via acanarypox virus-based vector, was used as the basis for a vaccine incats (Tartaglia et al., 1993, J. Virol. 67:2370-2375). The nucleotidesequence of the FeLV env gene (SEQ ID NO:19), the deduced amino acidsequence of its gene product Env, and neutralizing regions of Env havebeen disclosed previously (Stewart et al., 1986, J. Virol. 58:825-834;Elder et al., 1987, J. Virol. 61:8-15, respectively; the disclosures ofwhich are herein incorporated by reference). Cats immunized with thevaccine resisted subsequent challenge with FeLV, in the absence ofdetectable FeLV-neutralizing antibodies.

Using the methods of the present invention, as illustrated in Examples1-4, and 7, an expression cassette can be constructed to include anycombination of two or more genes, wherein one of the genes encodes theFeLV Env protein. Other exogenous genes that can be included in such anexpression cassette include genes that encode FIV Gag, FIV Env, FIPV M,FIPV N, FCV capsid protein, FPV VP2, or rabies-G. The expressioncassette may then be inserted into an insertion vector, and subsequentlyrecombined into the TK gene of raccoon poxvirus, thereby forming amultivalent recombinant raccoon poxvirus.

Alternatively, using the methods of the present invention, asillustrated in Examples 1-4, 6, and 7, an expression cassette can beconstructed to include any combination of two or more genes, wherein oneof the genes encodes FELV Env. Other exogenous genes that can beincluded in such an expression cassette include genes that encode FIVGag, FIV Env, FIPV M, FIPV N, FCV capsid protein, FPV VP2, or rabies-G.The expression cassette may then be inserted into an insertion vector,and subsequently recombined into the HA gene of raccoon poxvirus,thereby forming a multivalent recombinant raccoon poxvirus.

In another embodiment, using the methods according to the presentinvention, both the HA gene and the TK gene can be used as sites intowhich exogenous genes encoding feline pathogen antigens are recombinedinto raccoon poxvirus. Combinations of such exogenous genes includegenes which encode FeLV Env, FIV Gag, FIV Env, FIPV M, FIPV N, FCVcapsid protein, FPV VP2, and rabies-G. With all of these embodiments,using the methods according to Example 5 and other methods known in theart, vaccine stocks of recombinant raccoon poxvirus containing exogenousgenes encoding feline pathogen antigens can be prepared, andadministered in a vaccination process to felines.

It should be understood that while the invention has been described indetail herein, the examples were for illustrative purposes only. Othermodifications of the embodiments of the present invention that areobvious to those skilled in the art of molecular biology, veterinarymedicine, and related disciplines are intended to be within the scope ofthe appended claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 19(2) INFORMATION FOR SEQ ID NO:1 : (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 2254 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS:double-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii)HYPOTHETICAL: yes (iv) ORIGINAL SOURCE: (A) ORGANISM: felinepanleukopenia virus (v) FEATURE: (A) LOCATION: VP2 gene region,nucleotides 1 to 1752 (vi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG AGTGAT GGA GCA GTT CAA CCA GAC GGT GGT CAA CCT GCT GTC 45 Met Ser Asp GlyAla Val Gln Pro Asp Gly Gly Gln Pro Ala Val 1 5 10 15 AGA AAT GAA AGAGCT ACA GGA TCT GGG AAC GGG TCT GGA GGC GGG 90 Arg Asn Glu Arg Ala ThrGly Ser Gly Asn Gly Ser Gly Gly Gly 20 25 30 GGT GGT GGT GGT TCT GGG GGTGTG GGG ATT TCT ACG GGT ACT TTC 135 Gly Gly Gly Gly Ser Gly Gly Val GlyIle Ser Thr Gly Thr Phe 35 40 45 AAT AAT CAG ACG GAA TTT AAA TTT TTG GAAAAC GGA TGG GTG GAA 180 Asn Asn Gln Thr Glu Phe Lys Phe Leu Glu Asn GlyTrp Val Glu 50 55 60 ATC ACA GCA AAC TCA AGC AGA CTT GTA CAT TTA AAT ATGCCA GAA 225 Ile Thr Ala Asn Ser Ser Arg Leu Val His Leu Asn Met Pro Glu65 70 75 AGT GAA AAT TAT AAA AGA GTA GTT GTA AAT AAT ATG GAT AAA ACT 270Ser Glu Asn Tyr Lys Arg Val Val Val Asn Asn Met Asp Lys Thr 80 85 90 GCAGTT AAA GGA AAC ATG GCT TTA GAT GAC ACT CAT GTA CAA ATT 315 Ala Val LysGly Asn Met Ala Leu Asp Asp Thr His Val Gln Ile 95 100 105 GTA ACA CCTTGG TCA TTG GTT GAT GCA AAT GCT TGG GGA GTT TGG 360 Val Thr Pro Trp SerLeu Val Asp Ala Asn Ala Trp Gly Val Trp 110 115 120 TTT AAT CCA GGA GATTGG CAA CTA ATT GTT AAT ACT ATG AGT GAG 405 Phe Asn Pro Gly Asp Trp GlnLeu Ile Val Asn Thr Met Ser Glu 125 130 135 TTG CAT TTA GTT AGT TTT GAACAA GAA ATT TTT AAT GTT GTT TTA 450 Leu His Leu Val Ser Phe Glu Gln GluIle Phe Asn Val Val Leu 140 145 150 AAG ACT GTT TCA GAA TCT GCT ACT CAGCCA CCA ACT AAA GTT TAT 495 Lys Thr Val Ser Glu Ser Ala Thr Gln Pro ProThr Lys Val Tyr 155 160 165 AAT AAT GAT TTA ACT GCA TCA TTG ATG GTT GCATTA GAT AGT AAT 540 Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala Leu AspSer Asn 170 175 180 AAT ACT ATG CCA TTT ACT CCA GCA GCT ATG AGA TCT GAGACA TTG 585 Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg Ser Glu Thr Leu185 190 195 GGT TTT TAT CCA TGG AAA CCA ACC ATA CCA ACT CCA TGG AGA TAT630 Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp Arg Tyr 200 205210 TAT TTT CAA TGG GAT AGA ACA TTA ATA CCA TCT CAT ACT GGA ACT 675 TyrPhe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly Thr 215 220 225 AGTGGC ACA CCA ACA AAT ATA TAT CAT GGT ACA GAT CCA GAT GAT 720 Ser Gly ThrPro Thr Asn Ile Tyr His Gly Thr Asp Pro Asp Asp 230 235 240 GTT CAA TTTTAT ACT ATT GAA AAT TCT GTG CCA GTA CAC TTA CTA 765 Val Gln Phe Tyr ThrIle Glu Asn Ser Val Pro Val His Leu Leu 245 250 255 AGA ACA GGT GAT GAATTT GCT ACA GGA ACA TTT TTT TTT GAT TGT 810 Arg Thr Gly Asp Glu Phe AlaThr Gly Thr Phe Phe Phe Asp Cys 260 265 270 AAA CCA TGT AGA CTA ACA CATACA TGG CAA ACA AAC AGA GCA TTG 855 Lys Pro Cys Arg Leu Thr His Thr TrpGln Thr Asn Arg Ala Leu 275 280 285 GGC TTA CCA CCA TTT CTA AAT TCT TTGCCT CAA TCT GAA GGA GCT 900 Gly Leu Pro Pro Phe Leu Asn Ser Leu Pro GlnSer Glu Gly Ala 290 295 300 ACT AAC TTT GGT GAT ATA GGA GTT CAA CAA GATAAA AGA CGT GGT 945 Thr Asn Phe Gly Asp Ile Gly Val Gln Gln Asp Lys ArgArg Gly 305 310 315 GTA ACT CAA ATG GGA AAT ACA GAC TAT ATT ACT GAA GCTACT ATT 990 Val Thr Gln Met Gly Asn Thr Asp Tyr Ile Thr Glu Ala Thr Ile320 325 330 ATG AGA CCA GCT GAG GTT GGT TAT AGT GCA CCA TAT TAT TCT TTT1035 Met Arg Pro Ala Glu Val Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe 335 340345 GAA GCG TCT ACA CAA GGG CCA TTT AAA ATA CCT ATT GCA GCA GGA 1080 GluAla Ser Thr Gln Gly Pro Phe Lys Ile Pro Ile Ala Ala Gly 350 355 360 CGGGGG GGA GCG CAA ACA GAT GAA AAT CAA GCA GCA GAT GGT GAT 1125 Arg Gly GlyAla Gln Thr Asp Glu Asn Gln Ala Ala Asp Gly Asp 365 370 375 CCA AGA TATGCA TTT GGT AGA CAA CAT GGT CAA AAA ACT ACT ACA 1170 Pro Arg Tyr Ala PheGly Arg Gln His Gly Gln Lys Thr Thr Thr 380 385 390 ACA GGA GAA ACA CCTGAG AGA TTT ACA TAT ATA GCA CAT CAA GAT 1215 Thr Gly Glu Thr Pro Glu ArgPhe Thr Tyr Ile Ala His Gln Asp 395 400 405 ACA GGA AGA TAT CCA GCA GGAGAT TGG ATT CAA AAT ATT AAC TTT 1260 Thr Gly Arg Tyr Pro Ala Gly Asp TrpIle Gln Asn Ile Asn Phe 410 415 420 AAC CTT CCT GTA ACA AAT GAT AAT GTATTG CTA CCA ACA GAT CCA 1305 Asn Leu Pro Val Thr Asn Asp Asn Val Leu LeuPro Thr Asp Pro 425 430 435 ATT GGA GGT AAA ACA GGA ATC AAC TAT ACT AATATA TTT AAT ACT 1350 Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile PheAsn Thr 440 445 450 TAT GGT CCT TTA ACT GCA TTA AAT AAT GTA CCA CCA GTTTAT CCA 1395 Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val Pro Pro Val Tyr Pro455 460 465 AAT GGT CAA ATT TGG GAT AAA GAA TTT GAT ACT GAC TTA AAA CCA1440 Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys Pro 470 475480 AGA CTT CAT GTA AAT GCA CCA TTT GTT TGT CAA AAT AAT TGT CCT 1485 ArgLeu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro 485 490 495 GGTCAA TTA TTT GTA AAA GTT GCG CCT AAT TTA ACA AAT GAA TAT 1530 Gly Gln LeuPhe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr 500 505 510 GAT CCT GATGCA TCT GCT AAT ATG TCA AGA ATT GTA ACT TAC TCA 1575 Asp Pro Asp Ala SerAla Asn Met Ser Arg Ile Val Thr Tyr Ser 515 520 525 GAT TTT TGG TGG AAAGGT AAA TTA GTA TTT AAA GCT AAA CTA AGA 1620 Asp Phe Trp Trp Lys Gly LysLeu Val Phe Lys Ala Lys Leu Arg 530 535 540 GCA TCT CAT ACT TGG AAT CCAATT CAA CAA ATG AGT ATT AAT GTA 1665 Ala Ser His Thr Trp Asn Pro Ile GlnGln Met Ser Ile Asn Val 545 550 555 GAT AAC CAA TTT AAC TAT CTA CCA AATAAT ATT GGA GCT ATG AAA 1710 Asp Asn Gln Phe Asn Tyr Leu Pro Asn Asn IleGly Ala Met Lys 560 565 570 ATT GTA TAT GAA AAA TCT CAA CTA GCA CCT AGAAAA TTA TAT 1752 Ile Val Tyr Glu Lys Ser Gln Leu Ala Pro Arg Lys Leu Tyr575 580 TAATATACTT ACTATGTTTT TATGGTTATT ACATATCAAC TAGCACCTAG 1802AAAATTATAT TAATATACTT ACTATGTTTT TATGTTTATT ACATATTATT 1852 TTAAGATTAATTAAATTACA ACATAGAAAT ATTGTACTTG TATTTGATAT 1902 AGGATTTAGA AGGTTTGTTATATGGTATAC AATAACTGTA AGAAATAGAA 1952 GAACATTTAG ATCATGGTTA GTATGGTATACAATAACTGT AAGAAATAGA 2002 AGAACATTTA GATCATGGTT AGTAGTTTGT TTTATAAAATGTAATTGTAA 2052 ACTATTAATG TATGTTGTTA TGGTGTGGGT GGTTGGTTGG TTTGCCCTTA2102 GAATATGTTA AGGACCAAAA AAATCAATAA AAGACATTTA AAACTTAATG 2152GTCTCGTATA CTGTCTATAA GGTGAACTAA CCTTACCATA AGTATCAACT 2202 TGTCTTTAAGGGGGGGGTGG GTGGGAGATG CACAATATCA GTAGACTGAC 2252 TG 2254 (2) INFORMATIONFOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1575nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: double-stranded (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: yes (iv)ORIGINAL SOURCE: (A) ORGANISM: rabies virus (v) FEATURE: (A) LOCATION:rabies-G gene region, nucleotides 1 to 1572 (B) OTHER INFORMATION:mature protein begins with amino acid 20 (Lys) (vi) SEQUENCEDESCRIPTION: SEQ ID NO:2: ATG GTT CCT CAG GCT CTC CTG TTT GTA CCC CTTCTG GTT TTT 42 Met Val Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe 15 10 CCA TTG TGT TTT GGG AAA TTC CCT ATT TAC ACG ATA CTA GAC 84 Pro LeuCys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Leu Asp 15 20 25 AAG CTT GGT CCCTGG AGC CCG ATT GAC ATA CAT CAC CTC AGC 126 Lys Leu Gly Pro Trp Ser ProIle Asp Ile His His Leu Ser 30 35 40 TGC CCA AAC AAT TTG GTA GTG GAG GACGAA GGA TGC ACC AAC 168 Cys Pro Asn Asn Leu Val Val Glu Asp Glu Gly CysThr Asn 45 50 55 CTG TCA GGG TTC TCC TAC ATG GAA CTT AAA GTT GGA TAC ATC210 Leu Ser Gly Phe Ser Tyr Met Glu Leu Lys Val Gly Tyr Ile 60 65 70 TTAGCC ATA AAA ATG AAC GGG TTC ACT TGC ACA GGC GTT GTG 252 Leu Ala Ile LysMet Asn Gly Phe Thr Cys Thr Gly Val Val 75 80 ACG GAG GCT GAA ACC TACACT AAC TTC GTT GGT TAT GTC ACA 294 Thr Glu Ala Glu Thr Tyr Thr Asn PheVal Gly Tyr Val Thr 85 90 95 ACC ACG TTC AAA AGA AAG CAT TTC CGC CCA ACACCA GAT GCA 336 Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala100 105 110 TGT AGA GCC GCG TAC AAC TGG AAG ATG GCC GGT GAC CCC AGA 378Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg 115 120 125 TATGAA GAG TCT CTA CAC AAT CCG TAC CCT GAC TAC CGC TGG 420 Tyr Glu Glu SerLeu His Asn Pro Tyr Pro Asp Tyr Arg Trp 130 135 140 CTT CGA ACT GTA AAAACC ACC AAG GAG TCT CTC GTT ATC ATA 462 Leu Arg Thr Val Lys Thr Thr LysGlu Ser Leu Val Ile Ile 145 150 TCT CCA AGT GTA GCA GAT TTG GAC CCA TATGAC AGA TCC CTT 504 Ser Pro Ser Val Ala Asp Leu Asp Pro Tyr Asp Arg SerLeu 155 160 165 CAC TCG AGG GTC TTC CCT AGC GGG AAG TGC TCA GGA GTA GCG546 His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser Gly Val Ala 170 175 180GTG TCT TCT ACC TAC TGC TCC ACT AAC CAC GAT TAC ACC ATT 588 Val Ser SerThr Tyr Cys Ser Thr Asn His Asp Tyr Thr Ile 185 190 195 TGG ATG CCC GAGAAT CCG AGA CTA GGG ATG TCT TGT GAC ATT 630 Trp Met Pro Glu Asn Pro ArgLeu Gly Met Ser Cys Asp Ile 200 205 210 TTT ACC AAT AGT AGA GGG AAG AGAGCA TCC AAA GGG AGT GAG 672 Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser LysGly Ser Glu 215 220 ACT TGC GGC TTT GTA GAT GAA AGA GGC CTA TAT AAG TCTTTA 714 Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu 225 230235 AAA GGA GCA TGC AAA CTC AAG TTA TGT GGA GTT CTA GGA CTT 756 Lys GlyAla Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu 240 245 250 AGA CTT ATGGAT GGA ACA TGG GTC GCG ATG CAA ACA TCA AAT 798 Arg Leu Met Asp Gly ThrTrp Val Ala Met Gln Thr Ser Asn 255 260 265 GAA ACC AAA TGG TGC GCT CCCGAT CAG TTG GTG AAC CTG CAC 840 Glu Thr Lys Trp Cys Pro Pro Asp Gln LeuVal Asn Leu His 270 275 280 GAC TTT CGC TCA GAC GAA ATT GAG CAC CTT GTTGTA GAG GAG 882 Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val Val Glu Glu285 290 TTG GTC AGG AAG AGA GAG GAG TGT CTG GAT GCA CTA GAG TCC 924 LeuVal Arg Lsy Arg Glu Glu Cys Leu Asp Ala Leu Glu Ser 295 300 305 ATC ATGACA AAC AAG TCA GTG AGT TTC AGA CGT CTC AGT CAT 966 Ile Met Thr Thr LysSer Val Ser Phe Arg Arg Leu Ser His 310 315 320 TTA AGA AAA CTT GTC CCTGGG TTT GGA AAA GCA TAT ACC ATA 1008 Leu Arg Lys Leu Val Pro Gly Phe GlyLys Ala Tyr Thr Ile 325 330 335 TTC AAC AAG ACC TTG ATG GAA GCC GAT GCTCAC TAC AAG TCA 1050 Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr LysSer 340 345 350 GTC AGA ACT TGG AAT GAG ATC CTC CCT TCA AAA GGG TGT TTA1092 Val Arg Thr Trp Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu 355 360 AGAGTT GGG GGG AGG TGT CAT CCT CAT GTG AAC GGG GTG TTT 1134 Arg Val Gly GlyArg Cys His Pro His Val Asn Gly Val Phe 365 370 375 TTC AAT GGT ATA ATATTA GGA CCT GAC GGC AAT GTC TTA ATC 1176 Phe Asn Gly Ile Ile Leu Gly ProAsp Gly Asn Val Leu Ile 380 385 390 CCA GAG ATG CAA TCA TCC CTC CTC CAGCAA CAT ATG GAG TTG 1218 Pro Glu Met Gln Ser Ser Leu Leu Gln Gln His MetGlu Leu 395 400 405 TTG GAA TCC TCG GTT ATC CCC CTT GTG CAC CCC CTG GCAGAC 1260 Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala Asp 410 415420 CCG TCT ACC GTT TTC AAG GAC GGT GAC GAG GCT GAG GAT TTT 1302 Pro SerThr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe 425 430 GTT GAA GTT CACCTT CCC GAT GTG CAC AAT CAG GTC TCA GGA 1344 Val Glu Val His Leu Pro AspVal His Asn Gln Val Ser Gly 435 440 445 GTT GAC TTG GGT CTC CCG AAC TGGGGG AAG TAT GTA TTA CTG 1386 Val Asp Leu Gly Leu Pro Asn Trp Gly Lys TyrVal Leu Leu 450 455 460 AGT GCA GGG GCC CTG ACT GCC TTG ATG TTG ATA ATTTTC CTG 1428 Ser Ala Gly Ala Leu Thr Ala Leu Met Leu Ile Ile Phe Leu 465470 475 ATG ACA TGT TGT AGA AGA GTC AAT CGA TCA GAA CCT ACG CAA 1470 MetThr Cys Cys Arg Arg Val Asn Arg Ser Glu Pro Thr Gln 480 485 490 CAC AATCTC AGA GGG ACA GGG AGG GAG GTG TCA GTC ACT CCC 1512 His Asn Leu Arg GlyThr Gly Arg Glu Val Ser Val Thr Pro 495 500 CAA AGC GGG AAG ATC ATA TCTTCA TGG GAA TCA CAC AAG AGT 1554 Gln Ser Gly Lys Ile Ile Ser Ser Trp GluSer His Lys Ser 505 510 515 GGG GGT GAG ACC AGA CTG TGA 1575 Gly Gly GluThr Arg Leu 520 524 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 44 nucleotides (B) TYPE: nucleic acid (C)STRANDEDNESS: single-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (iii) HYPOTHETICAL: no (iv) FEATURE: P11 late promoter and leadersequence (v) SEQUENCE DESCRIPTION: SEQ ID NO:3: TAAAAATATA GTAGAATTTCATTTTGTTTT TTTCTATGCT ATAA 44 (2) INFORMATION FOR SEQ ID NO:4: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 nucleotides (B) TYPE: nucleicacid (C) STRANDEDNESS: single-stranded (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv) FEATURE: forward primer(v) SEQUENCE DESCRIPTION: SEQ ID NO:4: CGGGATCCAT TTTTCCTTCG TTTGCCAT 28(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 28 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS:single-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii)HYPOTHETICAL: no (iv) FEATURE: reverse primer (v) SEQUENCE DESCRIPTION:SEQ ID NO:5: CGGGTACCGA TTTCTCCGTG ATAGGTAT 28 (2) INFORMATION FOR SEQID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 nucleotides (B)TYPE: nucleic acid (C) STRANDEDNESS: single-stranded (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv) FEATURE:sequencing primer (v) SEQUENCE DESCRIPTION: SEQ ID NO:6: CTACTTGCATAGATAGGT 18 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 2007 nucleotides (B) TYPE: nucleic acid (C)STRANDEDNESS: double-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (iii) HYPOTHETICAL: yes (iv) ORIGINAL SOURCE: (A) ORGANISM: felinecalicivirus (v) FEATURE: (A) LOCATION: capsid protein gene region (vi)SEQUENCE DESCRIPTION: SEQ ID NO:7: ATG TGC TCA ACC TGC GCT AAC GTG CTTAAA TAC TAT GAT TGG GAT 45 Met Cys Ser Thr Cys Ala Asn Val Leu Lys TyrTyr Asp Trp Asp 1 5 10 15 CCT CAC ATC AAA TTG GTA ATC AAC CCC AAC AAATTT CTA CAT GTT 90 Pro His Ile Lys Leu Val Ile Asn Pro Asn Lys Phe LeuHis Val 20 25 30 GGC TTC TGC GAT AAC CCT TTA ATG TGT TGT TAT CCT GAA TTACTA 135 Gly Phe Cys Asp Asn Pro Leu Met Cys Cys Tyr Pro Glu Leu Leu 3540 45 CCT GAA TTT GGC ACC ATG TGG GAT TGT GAT CAA TCG CCA CTC CAA 180Pro Glu Phe Gly Thr Met Trp Asp Cys Asp Gln Ser Pro Leu Gln 50 55 60 GTCTAC CTT GAG TCA ATC CTG GGT GAT GAT GAA TGG TCC TCC ACT 225 Val Tyr LeuGlu Ser Ile Leu Gly Asp Asp Glu Trp Ser Ser Thr 65 70 75 CAT GAA GCA ATTGAC CCA GTT GTG CCA CCA ATG CAT TGG GAT GAA 270 His Glu Ala Ile Asp ProVal Val Pro Pro Met His Trp Asp Glu 80 85 90 GCC GGA AAA ATC TTC CAA CCACAC CCT GGC GTC CTT ATG CAT CAC 315 Ala Gly Lys Ile Phe Gln Pro His ProGly Val Leu Met His His 95 100 105 CTC ATC TGT AAG GTT GCA GAA GGA TGGGAC CCA AAC CTG CCA CTT 360 Leu Ile Cys Lys Val Ala Glu Gly Trp Asp ProAsn Leu Pro Leu 110 115 120 TTC CGC TTG GAA GCG GAC GAT GGT TCC ATC ACGACA CCT GAA CAG 405 Phe Arg Leu Glu Ala Asp Asp Gly Ser Ile Thr Thr ProGlu Gln 125 130 135 GGA ACA ATG GTT GGT GGA GTC ATT GCT GAG CCC AAC GCCCAA ATG 450 Gly Thr Met Val Gly Gly Val Ile Ala Glu Pro Asn Ala Gln Met140 145 150 TCA ACC GCA GCT GAC ATG GCC ACT GGG AAA AGT GTG GAC TCT GAG495 Ser Thr Ala Ala Asp Met Ala Thr Gly Lys Ser Val Asp Ser Glu 155 160165 TGG GAA GCC TTC TTC TCC TTT CAC ACT AGT GTG AAC TGG AGC ACA 540 TrpGlu Ala Phe Phe Ser Phe His Thr Ser Val Asn Trp Ser Thr 170 175 180 TCTGAA ACT CAG GGG AAG ATA CTC TTT AAA CAA TCC TTA GGA CCA 585 Ser Glu ThrGln Gly Lys Ile Leu Phe Lys Gln Ser Leu Gly Pro 185 190 195 TTG CTC AACCCC TAC CTT ACC CAT CTT GCA AAG CTG TAT GTT GCT 630 Leu Leu Asn Pro TyrLeu Thr His Leu Ala Lys Leu Tyr Val Ala 200 205 210 TGG TCT GGT TCT GTTGAT GTT AGG TTT TCT ATT TCT GGA TCT GGT 675 Trp Ser Gly Ser Val Asp ValArg Phe Ser Ile Ser Gly Ser Gly 215 220 225 GTC TTT GGA GGG AAA TTA GCTGCT ATT GTT GTG CCG CCA GGA ATT 720 Val Phe Gly Gly Lys Leu Ala Ala IleVal Val Pro Pro Gly Ile 230 235 240 GAT CCT GTT CAA AGT ACT TCA ATG CTGCAA TAT CCT CAT GTC CTC 765 Asp Pro Val Gln Ser Thr Ser Met Leu Gln TyrPro His Val Leu 245 250 255 TTT GAT GCT CGT CAA GTT GAA CCT GTT ATC TTTTCC ATT CCC GAT 810 Phe Asp Ala Arg Gln Val Glu Pro Val Ile Phe Ser IlePro Asp 260 265 270 CTA AGA AGC ACC TTA TAT CAC CTT ATG TCT GAC ACT GATACC ACA 855 Leu Arg Ser Thr Leu Tyr His Leu Met Ser Asp Thr Asp Thr Thr275 280 285 TCG TTG GTA ATC ATG GTG TAC AAT GAT CTT ATT AAC CCC TAT GCT900 Ser Leu Val Ile Met Val Tyr Asn Asp Leu Ile Asn Pro Tyr Ala 290 295300 AAT GAC TCA AAC TCT TCG GGC TGC ATT GTC ACT GTG GAA ACT AAA 945 AsnAsp Ser Asn Ser Ser Gly Cys Ile Val Thr Val Glu Thr Lys 305 310 315 CCGGGG CCA GAT TTC AAG TTT CAC CTC TTA AAA CCT CCT GGG TCT 990 Pro Gly ProAsp Phe Lys Phe His Leu Leu Lys Pro Pro Gly Ser 320 325 330 ATG TTA ACTCAC GGA TCT ATC CCA TCT GAT CTA ATC CCA AAA TCA 1035 Met Leu Thr His GlySer Ile Pro Ser Asp Leu Ile Pro Lys Ser 335 340 345 TCT TCG CTT TGG ATTGGA AAT CGG TTT TGG TCT GAC ATA ACC GAT 1080 Ser Ser Leu Trp Ile Gly AsnArg Phe Trp Ser Asp Ile Thr Asp 350 355 360 TTT GTA ATT CGG CCT TTT GTGTTC CAG GCA AAT CGA CAC TTT GAT 1125 Phe Val Ile Arg Pro Phe Val Phe GlnAla Asn Arg His Phe Asp 365 370 375 TTC AAC CAA GAG ACA GCA GGT TGG AGCACC CCA AGG TTT CGC CCA 1170 Phe Asn Gln Glu Thr Ala Gly Trp Ser Thr ProArg Phe Arg Pro 380 385 390 ATT ACT ATC ACT ATC AGT GTT AAG GAG TCA GCAAAG CTT GGT ATT 1215 Ile Thr Ile Thr Ile Ser Val Lys Glu Ser Ala Lys LeuGly Ile 395 400 405 GGA GTG GCC ACC GAC TAC ATT GTT CCC GGC ATA CCA GATGGA TGG 1260 Gly Val Ala Thr Asp Tyr Ile Val Pro Gly Ile Pro Asp Gly Trp410 415 420 CCC GAC ACA ACA ATC CCA GGT GAG TTG GTA CCT GTT GGT GAC TAT1305 Pro Asp Thr Thr Ile Pro Gly Glu Leu Val Pro Val Gly Asp Tyr 425 430435 GCC ATC ACT AAT GGC ACC AAC AAT GAT ATC ACC ACA GCT GCG CAG 1350 AlaIle Thr Asn Gly Thr Asn Asn Asp Ile Thr Thr Ala Ala Gln 440 445 450 TACGAT GCA GCC ACT GAG ATT AGA AAC AAC ACC AAT TTC AGA GGC 1395 Tyr Asp AlaAla Thr Glu Ile Arg Asn Asn Thr Asn Phe Arg Gly 455 460 465 ATG TAC ATTTGT GGT TCT CTT CAA AGA GCT TGG GGG GAT AAG AAG 1440 Met Tyr Ile Cys GlySer Leu Gln Arg Ala Trp Gly Asp Lys Lys 470 475 480 ATT TCA AAT ACT GCTTTT ATC ACA ACC GGC ACG GTT GAT GGA GCC 1485 Ile Ser Asn Thr Ala Phe IleThr Thr Gly Thr Val Asp Gly Ala 485 490 495 AAA TTG ATA CCC AGT AAT ACCATT GAC CAA ACA AAA ATT GCC GTA 1530 Lys Leu Ile Pro Ser Asn Thr Ile AspGln Thr Lys Ile Ala Val 500 505 510 TTC CAA GAC ACA CAT GCG AAT AAG CATGTC CAG ACC TCG GAC GAC 1575 Phe Gln Asp Thr His Ala Asn Lys His Val GlnThr Ser Asp Asp 515 520 525 ACA TTG GCC CTG CTT GGT TAT ACT GGT ATT GGTGAG GAA GCA ATT 1620 Thr Leu Ala Leu Leu Gly Tyr Thr Gly Ile Gly Glu GluAla Ile 530 535 540 GGT GCT GAC CGC GAT AGA GTT GTG CGA ATT AGC GTC CTCCCG GAA 1665 Gly Ala Asp Arg Asp Arg Val Val Arg Ile Ser Val Leu Pro Glu545 550 555 CGT GGC GCA CGT GGT GGC AAT CAC CCA ATC TTC CAC AAA AAC TCT1710 Arg Gly Ala Arg Gly Gly Asn His Pro Ile Phe His Lys Asn Ser 560 565570 ATC AAG CTT GGT TAT GTA ATT AGG TCC ATT GAT GTG TTC AAT TCT 1755 IleLys Leu Gly Tyr Val Ile Arg Ser Ile Asp Val Phe Asn Ser 575 580 585 CAAATT CTG CAT ACC TCT AGG CAA CTT TCC CTC AAT CAT TAC TTA 1800 Gln Ile LeuHis Thr Ser Arg Gln Leu Ser Leu Asn His Tyr Leu 590 595 600 TTG TCG CCTGAC TCC TTT GCT GTC TAT AGG ATT ATT GAC TCT AAT 1845 Leu Ser Pro Asp SerPhe Ala Val Tyr Arg Ile Ile Asp Ser Asn 605 610 615 GGA TCC TGG TTT GACATA GGC ATT GAT AAT GAT GGA TTT TCT TTT 1890 Gly Ser Trp Phe Asp Ile GlyIle Asp Asn Asp Gly Phe Ser Phe 620 625 630 GTT GGT GTA TCA AGT ATT GGTAAA TTA GAG TTT CCT TTA ACT GCC 1935 Val Gly Val Ser Ser Ile Gly Lys LeuGlu Phe Pro Leu Thr Ala 635 640 645 TCC TAC ATG GGA ATT CAA TTG GCA AAAATT CGA CTT GCC TCT AAC 1980 Ser Tyr Met Gly Ile Gln Leu Ala Lys Ile ArgLeu Ala Ser Asn 650 655 660 ATT AGG AGT GTG ATG ACA AAA TTA TGA 2007 IleArg Ser Val Met Thr Lys Leu 665 (2) INFORMATION FOR SEQ ID NO:8: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 582 nucleotides (B) TYPE: nucleicacid (C) STRANDEDNESS: double-stranded (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv) FEATURE: HA left arm (v)SEQUENCE DESCRIPTION: SEQ ID NO:8: ATTAAACGCA AATATCCATG GAAAACGCGCAGTATACAGA CGATTTTTTA 50 CAGTATTTGG AGAGTTTTAT AGGAAGTATA TAGAGTAGAACCAGAATTTT 100 GTAAAAATAA ATCACATTTT TATACTAATA TGAAACAACT ATCGATAGTT150 ATATTGCTAC TATCGATAGT ATATACAACC AAACCTCATC CTACACAGAT 200ATCAAAAAAA CTAGGCGATG ATGCTACTCT ATCGTGTAAT AGAAACAATA 250 CACATGGATATCTTGTCATG AGTTCTTGGT ATAAGAAACC AGACTCCATT 300 ATTCTCTTAG CAGCCAAAAACGATGTCGTA TACTTTGATG ATTATACAGC 350 GGATAAAGTA TCATACGATT CACCGTATGATACTCTAGCT ACAATTATTA 400 CAATTAAATC ATTGACATCT GCAGATGCAG GTACTTATATATGCGCATTC 450 TTTATAACAT CAACAAATGA TACGGATAAA ATAGATTATG AAGAATACTT500 CATAGATTTG GTTGTAAATC CAGCTAATGT ATCCACTATT GACGCGATTC 550TATCAGGATC TAATTTCTCC GTGATAGGTA TC 582 (2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 447 nucleotides (B) TYPE:nucleic acid (C) STRANDEDNESS: double-stranded (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv) FEATURE: HA right arm (v)SEQUENCE DESCRIPTION: SEQ ID NO:9: CTCTAGCGCC TAACCCCAGG CGACCGACGACAACCTTTAT GATACATATA 50 ATGAACCAAT ATCTGTATCA TCCTCGATAC CAACAACGGTAGAAAGTGTT 100 ACAATATCTA CTACAAAATA TACAACTAGT GACTTTATAG AGATATTTGG150 CATTGTTTCA CTAATTTTAT TATTGGCCGT GGCGATTTTC TGTATTATAT 200TATTTCTGTA GTGGACGGTC TCGTAAACAA GAAACAAATA TATTATAGAT 250 TTTAACTCAGATAAATGTCT GGAATAATTA AATCTATCGT TTTGAGCGGA 300 CCATCTGGTT CCGGCAAGACAGCTATAGTC AGGAGACTCT TACAAGATTA 350 TGGAAATATA TTTGGATTTG TGGTATCCCATACCACTAGA TTTCCTCGTC 400 CTATGGAACG AGAAGGTGTC GACTACCATT ACGTTAACAGAGAGGCC 447 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 40 nucleotides (B) TYPE: nucleic acid (C)STRANDEDNESS: single-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (iii) HYPOTHETICAL: no (iv) FEATURE: primer P3 (v) SEQUENCEDESCRIPTION: SEQ ID NO:10: GATACCTATC ACGGAGAAAT TAGATCCTGA TAGAATCGCG40 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 22 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS:single-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii)HYPOTHETICAL: no (iv) FEATURE: primer P1 (v) SEQUENCE DESCRIPTION: SEQID NO:11: ATTAAACGCA AATATCCATG GG 22 (2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 nucleotides (B) TYPE:nucleic acid (C) STRANDEDNESS: single-stranded (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv) FEATURE: primer F2 (v)SEQUENCE DESCRIPTION: SEQ ID NO:12: GCGGTACCCT GGGGTTAGGC GATAGAG 27 (2)INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:20 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single-stranded(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv)FEATURE: primer P5 (v) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATTTCTCCGTGATAGGTATC 20 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 22 nucleotides (B) TYPE: nucleic acid (C)STRANDEDNESS: single-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (iii) HYPOTHETICAL: no (iv) FEATURE: primer P2 (v) SEQUENCEDESCRIPTION: SEQ ID NO:14: GGCCTCTCTG TTAACGTAAT GG 22 (2) INFORMATIONFOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS: single-stranded (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: no (iv)FEATURE: primer F1 (v) SEQUENCE DESCRIPTION: SEQ ID NO:15: GCGTCGAAGTTTGAGCATGT GC 22 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 40 nucleotides (B) TYPE: nucleic acid (C)STRANDEDNESS: single-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (iii) HYPOTHETICAL: no (iv) FEATURE: primer P4 (v) SEQUENCEDESCRIPTION: SEQ ID NO:16: CTCTAGCGCC TAACCCCAGG CGACCGACGA CAACCTTTAT40 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 840 nucleotides (B) TYPE: nucleic acid (C) STRANDEDNESS:double-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (iii)HYPOTHETICAL: yes (iv) ORIGINAL SOURCE: (A) ORGANISM: feline infectiousperitonitis virus (v) FEATURE: (A) LOCATION: membrane glycoprotein generegion (vi) SEQUENCE DESCRIPTION: SEQ ID NO:17: AAACCAAGGC ATATAATCCCGACGAAGCAT TTTTGGTTTG AACTAAACAA A 51 ATG AAG TAC ATT TTG CTA ATA CTCGCG TGC ATA ATT GCA TGC GTT 96 Met Lys Tyr Ile Leu Leu Ile Leu Ala CysIle Ile Ala Cys Val 1 5 10 15 TAT GGT GAA CGC TAC TGT GCC ATG CAA GACAGT GGC TTG CAG TGT 141 Tyr Gly Glu Arg Tyr Cys Ala Met Gln Asp Ser GlyLeu Gln Cys 20 25 30 ATT AAT GGC ACA AAT TCA AGA TGT CAA ACC TGC TTT GAACGT GGT 186 Ile Gln Gly Thr Gln Ser Arg Cys Gln Thr Cys Phe Glu Arg Gly35 40 45 GAT CTT ATT TGG CAT CTT GCT AAC TGG AAC TTC AGC TGG TCT GTA 231Asp Leu Ile Trp His Leu Ala Asn Trp Asn Phe Ser Trp Ser Val 50 55 60 ATATTG ATT GTT TTT ATA ACA GTG TTA CAA TAT GGC AGA CCA CAA 276 Ile Leu IleVal Phe Ile Thr Val Leu Gln Tyr Gly Arg Pro Gln 65 70 75 TTT AGC TGG CTCGTT TAT GGC ATT AAA ATG CTG ATC ATG TGG CTA 321 Phe Ser Trp Leu Val TyrGly Ile Lys Met Leu Ile Met Trp Leu 80 85 90 TTA TGG CCT ATT GTT CTA GCGCTT ACG ATT TTT AAT GCA TAC TCT 366 Leu Trp Pro Ile Val Leu Ala Leu ThrIle Phe Asn Ala Tyr Ser 95 100 105 GAG TAC CAA GTT TCC AGA TAT GTA ATGTTC GGC TTT AGT GTT GCA 411 Glu Tyr Gln Val Ser Arg Tyr Val Met Phe GlyPhe Ser Val Ala 110 115 120 GGT GCA GTT GTA ACG TTT GCA CTT TGG ATG ATGTAT TTT GTG AGA 456 Gly Ala Val Val Thr Phe Ala Leu Trp Met Met Tyr PheVal Arg 125 130 135 TCT GTT CAG CTA TAT AGA AGA ACC AAA TCA TGG TGG TCTTTT AAT 501 Ser Val Gln Leu Tyr Arg Arg Thr Lys Ser Trp Trp Ser Phe Asn140 145 150 CCT GAG ACT AAT GCA ATT CTT TGT GTT AAT GCA TTG GGT AGA AGT546 Pro Glu Thr Asn Ala Ile Leu Cys Val Asn Ala Leu Gly Arg Ser 155 160165 TAT GTG CTT CCC TTA GAT GGT ACT CCT ACA GGT GTT ACC CTT ACT 591 TyrVal Leu Pro Leu Asp Gly Thr Pro Thr Gly Val Thr Leu Thr 170 175 180 CTACTT TCA GGA AAT CTA TAT GCT GAA GGT TTC AAA ATG GCT GGT 636 Leu Leu SerGly Asn Leu Tyr Ala Glu Gly Phe Lys Met Ala Gly 185 190 195 GGT TTA ACCATC GAG CAT TTG CCT AAA TAC GTC ATG ATT GCT ACA 681 Gly Leu Thr Ile GluHis Leu Pro Lys Tyr Val Met Ile Ala Thr 200 205 210 CCT AGT AGA ACC ATCGTT TAT ACA TTA GTT GGA AAA CAA TTA AAA 726 Pro Ser Arg Thr Ile Val TyrThr Ile Val Gly Lys Gln Leu Lys 215 220 225 GCA ACT ACT GCC ACA GGA TGGGCT TAC TAC GTA AAA TCT AAA GCT 771 Ala Thr Thr Ala Thr Gly Trp Ala TyrTyr Val Lys Ser Lys Ala 230 235 240 GGT GAT TAC TCA ACA GAA GCA CGT ACTGAC AAT TTG AGT GAA CAT 816 Gly Asp Tyr Ser Thr Glu Ala Arg Thr Asp AsnLeu Ser Glu His 245 250 255 GAA AAA TTA TTA CAT ATG GTG TAA 840 Glu LysLeu Leu His Met Val 260 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1144 nucleotides (B) TYPE: nucleic acid (C)STRANDEDNESS: double-stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (iii) HYPOTHETICAL: yes (iv) ORIGINAL SOURCE: (A) ORGANISM: felineinfectious peritonitis virus (v) FEATURE: (A) LOCATION: nucleocapsidprotein gene region (vi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ATG GCC ACACAG GGA CAA CGC GTC AAC TGG GGA GAT GAA CCT TCC 45 Met Ala Thr Gln GlyGln Arg Val Asn Trp Gly Asp Glu Pro Ser 1 5 10 15 AAA AGA CGT GGT CGTTCT AAC TCT CGT GGT CGG AAG AAT AAT GAT 90 Lys Arg Arg Gly Arg Ser AsnSer Arg Gly Arg Lys Asn Asn Asp 20 25 30 ATA CCT TTG TCA TTC TAC AAC CCCATT ACC CTC GAA CAA GGA TCT 135 Ile Pro Leu Ser Phe Tyr Asn Phe Ile ThrLeu Glu Gln Glu Ser 35 40 45 AAA TTT TGG AAT TTA TGT CCG AGA GAC CTT GTTCCC AAA GGA ATA 180 Lys Phe Trp Asn Leu Cys Pro Arg Asp Leu Val Pro LysGly Ile 50 55 60 GGT AAT AAG GAT CAA CAA ATT GGT TAT TGG AAT AGA CAG ATTCGT 225 Gly Asn Lys Asp Gln Gln Ile Gly Tyr Trp Asn Arg Gln Ile Arg 6570 75 TAT CGT ATT GTA AAA GGC CAG CGT AAG GAA CTC GCT GAG AGG TGG 270Tyr Arg Ile Val Lys Gly Gln Arg Lys Glu Leu Ala Glu Arg Trp 80 85 90 TTCTTT TAC TTC TTA GGT ACA GGA CCT CAT GCT GAT GCT AAA TTC 315 Phe Phe TyrPhe Leu Gly Thr Gly Phe His Ala Asp Ala Lys Phe 95 100 105 AAA GAC AAGATT GAT GGA GTC TTC TGG GTT GCA AGG GAT GGT GCC 360 Lys Asp Lys Ile AspGly Val Phe Trp Val Ala Arg Asp Gly Ala 110 115 120 ATG AAC AAG CCC ACAACG CTT GGC ACT CGT GGA ACC AAT AAC GAA 405 Met Asn Lys Pro Thr Thr LeuGly Thr Arg Gly Thr Asn Asn Glu 125 130 135 TCC AAA CCA CTG AGA TTT GATGGT AAG ATA CCG CCA CAG TTT CAG 450 Ser Lys Pro Leu Arg Phe Asp Gly LysIle Pro Pro Gln Phe Gln 140 145 150 CTT GAA GTG AAC CGT TCT AGG AAC AATTCA AGG TCT GGT TCT CAG 495 Leu Glu Val Asn Arg Ser Arg Asn Asn Ser ArgSer Gly Ser Gln 155 160 165 TCT AGA TCT GTT TCA AGA AAC AGA TCT CAA TCTAGA GGA AGA CAC 540 Ser Arg Ser Val Ser Arg Asn Arg Ser Gln Ser Arg GlyArg His 170 175 180 CAT TCC AAT AAC CAG AAT AAT AAT GTT GAG GAT ACA ATTGTA GCC 585 His Ser Asn Asn Gln Asn Asn Asn Val Glu Asp Thr Ile Val Ala185 190 195 GTG CTT GAA AAA TTA GGT GTT ACT GAC AAA CAA AGG TCA CGT TCT630 Val Leu Glu Lys Leu Gly Val Thr Asp Lys Gln Arg Ser Arg Ser 200 205210 AAA CCT AGA GAA CGT AGT GAT TCC AAA CCT AGG GAC ACA ACA CCT 675 LysPro Arg Glu Arg Ser Asp Ser Lys Pro Arg Asp Thr Thr Pro 215 220 225 AAGAAT GCC AAC AAA CAC ACC TGG AAG AAA ACT GCA GGC AAG GGA 720 Lys Asn AlaAsn Lys His Thr Trp Lys Lys Thr Ala Gly Lys Gly 230 235 240 GAT GTG ACAACT TTC TAT GGT GCT AGA AGT AGT TCA GCT AAC TTT 765 Asp Val Thr Thr PheTyr Gly Ala Arg Ser Ser Ser Ala Asn Phe 245 250 255 GGT GAT AGT GAT CTCGTT GCC AAT GGT AAC GCT GCC AAA TGC TAC 810 Gly Asp Ser Asp Leu Val AlaAsn Gly Asn Ala Ala Lys Cys Tyr 260 265 270 CCT CAG ATA GCT GAA TGT GTTCCA TCA GTG TCT AGC ATA ATC TTT 855 Pro Gln Ile Ala Glu Cys Val Pro SerVal Ser Ser Ile Ile Phe 275 280 285 GGC AGT CAA TGG TCT GCT GAA GAA GCTGGT GAT CAA GTG AAA GTC 900 Gly Ser Gln Trp Ser Ala Glu Glu Ala Gly AspGln Val Lys Val 290 295 300 ACG CTC ACT CAC ACC TAC TAC CTG CCA AAG GATGAT GCC AAA ACT 945 Thr Leu Thr His Thr Tyr Tyr Leu Pro Lys Asp Asp AlaLys Thr 305 310 315 AGT CAA TTC CTA GAA CAG ATT GAC GCT TAC AAG CGA CCTTCT GAA 990 Ser Gln Phe Leu Glu Gln Ile Asp Ala Tyr Lys Atg Pro Ser Glu320 325 330 GTG GCT AAG GAT CAG AGG CAA AGA AGA TCC CGT TCT AAG TCT GCT1035 Val Ala Lys Asp Gln Arg Gln Arg Arg Ser Arg Ser Lys Ser Ala 335 340345 GAT AAG AAG CCT GAG GAG TTG TCT GTA ACT CTT GTG GAG GCA TAC 1080 AspLys Lys Pro Glu Glu Lys Ser Val Thr Leu Val Glu Ala Tyr 350 355 360 ACAGAT GTG TTT GAT GAC ACA CAG GTT GAG ATG ATT GAT GAG GTT 1125 Thr Asp ValPhe Asp Asp Thr Gln Val Glu Met Ile Asp Glu Val 365 370 375 ACG AAC TAAACGCATGCTC 1144 Thr Asn 377 (2) INFORMATION FOR SEQ ID NO:19: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1979 nucleotides (B) TYPE: nucleicacid (C) STRANDEDNESS: double-stranded (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (iii) HYPOTHETICAL: yes (iv) ORIGINAL SOURCE: (A)ORGANISM: feline leukemia virus (v) FEATURE: (A) LOCATION: env generegion (vi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ACCACCAATC AAGACCTCTCGGACAGCCCC AGCTCAGACG ATCCATCAAG 50 ATG GAA AGT CCA ACG CAC CCA AAA CCCTCT AAA GAT AAG ACT CTC 95 Met Glu Ser Pro Thr His Pro Lys Pro Ser LysAsp Lys Thr Leu 1 5 10 15 TCG TGG AAC TTA GCG TTT CTG GTG GGG ATC TTATTT ACA ATA GAC 140 Ser Trp Asn Leu Ala Phe Leu Val Gly Ile Leu Phe ThrIle Asp 20 25 30 ATA GGA ATG GCC AAT CCT AGT CCA CAC CAA ATA TAT AAT GTAACT 185 Ile Gly Met Ala Asn Pro Ser Pro His Gln Ile Tyr Asn Val Thr 3540 45 TGG GTA ATA ACC AAT GTA CAA ACT AAC ACC CAA GCT AAC GCC ACC 230Trp Val Ile Thr Asn Val Gln Thr Asn Thr Gln Ala Asn Ala Thr 50 55 60 TCTATG TTA GGA ACC TTA ACC GAT GCC TAC CCT ACC CTA CAT GTT 275 Ser Met LeuGly Thr Leu Thr Asp Ala Tyr Pro Thr Leu His Val 65 70 75 GAC TTA TGT GACCTA GTG GGA GAC ACC TGG GAA CCT ATA GTC CTA 320 Asp Leu Cys Asp Leu ValGly Asp Thr Trp Glu Pro Ile Val Leu 80 85 90 AAC CCA ACC AAT GTA AAA CACGGG GCA CGT TAC TCC TCC TCA AAA 365 Asn Pro Thr Asn Val Lys His Gly AlaArg Tyr Ser Ser Ser Lys 95 100 105 TAT GGA TGT AAA ACT ACA GAT AGA AAAAAA CAG CAA CAG ACA TAC 410 Tyr Gly Cys Lys Thr Thr Asp Arg Lys Lys GlnGln Gln Thr Tyr 110 115 120 CCC TTT TAC GTC TGC CCC GGA CAT GCC CCC TCGTTG GGG CCA AAG 455 Pro Phe Tyr Val Cys Pro Gly His Ala Pro Ser Leu GlyPro Lys 125 130 135 GGA ACA CAT TGT GGA GGG GCA CAA GAT GGG TTT TGT GCCGCA TGG 500 Gly Thr His Cys Gly Gly Ala Gln Asp Gly Phe Cys Ala Ala Trp140 145 150 GGA TGT GAG ACC ACC GGA GAA GCT TGG TGG AAG CCC ACC TCC TCA545 Gly Cys Glu Thr Thr Gly Glu Thr Trp Trp Lys Pro Thr Ser Ser 155 160165 TGG GAC TAT ATC ACA GTA AAA AGA GGG AGT AGT CAG GAC AAT AGC 590 TrpAsp Tyr Ile Thr Val Lys Arg Gly Ser Ser Gln Asp Asn Ser 170 175 180 TGTGAG GGA AAA TGC AAC CCC CTG GTT TTG CAG TTC ACC CAG AAG 635 Cys Glu GlyLys Cys Asn Pro Leu Val Leu Gln Phe Thr Gln Lys 185 190 195 GGA AGA CAAGCC TCT TGG GAC GGA CCT AAG ATG TGG GGA TTG CGA 680 Gly Arg Gln Ala SerTrp Asp Gly Pro Lys Met Trp Gly Leu Arg 200 205 210 CTA TAC CGT ACA GGATAT GAC CCT ATC GCT TTA TTC ACG GTG TCC 725 Leu Tyr Arg Thr Gly Tyr AspPro Ile Ala Leu Phe Thr Val Ser 215 220 225 CGG CAG GTA TCA ACC ATT ACGCCG CCT CAG GCA ATG GGA CCA AAC 770 Arg Gln Val Ser Thr Ile Thr Pro ProGln Ala Met Gly Pro Asn 230 235 240 CTA GTC TTA CCT GAT CAA AAA CCC CCATCC CGA CAA TCT CAA ACA 815 Leu Val Leu Pro Asp Gln Lys Pro Pro Ser ArgGln Ser Gln Thr 245 250 255 GGG TCC AAA GTG GCG ACC CAG AGG CCC CAA ACGAAT GAA AGC GCC 860 Gly Ser Lys Val Ala Thr Gln Arg Pro Gln Thr Asn GluSer Ala 260 265 270 CCA AGG TCT GTT GCC CCC ACC ACC ATG GGT CCC AAA CGGATT GGG 905 Pro Arg Ser Val Ala Pro Thr Thr Met Gly Pro Lys Arg Ile Gly275 280 285 ACC GGA GAT AGG TTA ATA AAT TTA GTA CAA GGG ACA TAC CTA GCC950 Thr Gly Asp Arg Leu Ile Asn Leu Val Gln Gly Thr Tyr Leu Ala 290 295300 TTA AAT GCC ACC GAC CCC AAC AAA ACT AAA GAC TGT TGG CTC TGC 995 LeuAsn Ala Thr Asp Pro Asn Lys Thr Lys Asp Cys Trp Leu Cys 305 310 315 CTGGTT TCT CGA CCA CCC TAT TAC GAA GGG ATT GCA ATC TTA GGT 1040 Leu Val SerArg Pro Pro Tyr Tyr Glu Gly Ile Ala Ile Leu Gly 320 325 330 ACC TAC AGCAAC CAA ACA AAC CCC CCC CCA TCC TGC CTA TCT ACT 1085 Asn Tyr Ser Asn GlnThr Asn Pro Pro Pro Ser Cys Leu Ser Ile 335 340 345 CCG CAA CAC AAA CTAACT ATA TCT GAA GTA TCA GGG CAA GGA ATG 1130 Pro Gln His Lys Leu Thr IleSer Glu Val Ser Gly Gln Gly Met 350 355 360 TGC ATA GGG ACT GTT CCT AAAACC CAC CAG GCT TTG TGC AAT AAG 1175 Cys Ile Gly Thr Val Pro Lys Thr HisGln Ala Leu Cys Asn Lys 365 370 375 ACA CAA CAG GGA CAT ACA GGG GCG CACTAT CTA GCC GCC CCC AAC 1220 Thr Gln Gln Gly His Thr Gly Ala His Tyr LeuAla Ala Pro Asn 380 385 390 GGC ACC TAT TGG GCC TGT AAC ACT GGA CTC ACCCCA TGC ATT TCC 1265 Gly Thr Tyr Trp Ala Cys Asn Thr Gly Leu Thr Pro CysIle Ser 395 400 405 ATG GCG GTG CTC AAT TGG ACC TCT GAT TTT TGT GTC TTAATC GAA 1310 Met Ala Val Leu Asn Trp Thr Ser Asp Phe Cys Val Leu Ile Glu410 415 420 TTA TGG CCC AGA GTG ACT TAC CAT CAA CCC GAA TAT GTG TAC ACA1355 Leu Trp Pro Arg Val Thr Tyr His Gln Pro Glu Tyr Val Tyr Thr 425 430435 CAT TTT GCC AAA GCT GTC AGG TTC CGA AGA GAA CCA ATA TCA CTA 1400 HisPhe Ala Lys Ala Val Arg Phe Arg Arg Glu Pro Ile Ser Leu 440 445 450 ACGGTT GCC CTT ATG TTG GGA GGA CTT ACT GTA GGG GGC ATA GCC 1445 Thr Val AlaLeu Met Leu Gly Gly Leu Thr Val Gly Gly Ile Ala 455 460 465 GCG GGG GTCGGA ACA GGG ACT AAA GCC CTC CTT GAA ACA GCC CAG 1490 Ala Gly Val Gly ThrGly Thr Lys Ala Leu Leu Glu Thr Ala Gln 470 475 480 TTC AGA CAA CTA CAAATG GCC ATG CAC ACA GAC ATC CAG GCC CTA 1535 Phe Arg Gln Leu Gln Met AlaMet His Thr Asp Ile Gln Ala Leu 485 490 495 GAA GAA TCA ATT AGT GCC TTAGAA AAG TCC CTG ACC TCC CTT TCT 1580 Glu Glu Ser Ile Ser Ala Leu Glu LysSer Leu Thr Ser Leu Ser 500 505 510 GAA GTA GTC TTA CAA AAC AGA CGG GGCCTA GAT ATT CTA TTC TTA 1625 Glu Val Val Leu Gln Asn Arg Arg Glu Leu AspIle Leu Phe Leu 515 520 525 CAA GAG GGA GGG CTC TGT GCC GCA TTG AAA GAAGAA TGT TGC TTC 1670 Gln Glu Gly Gly Leu Cys Ala Ala Leu Lys Glu Glu CysCys Phe 530 535 540 TAT GCG GAT CAC ACC GGA CTC GTC CGA GAC AAT ATG GCCAAA TTA 1715 Tyr Ala Asp His Thr Gly Leu Val Arg Asp Asn Met Ala Lys Leu545 550 555 AGA GAA AGA CTA AAA CAG CGG CAA CAA CTG TTT GAC TCC CAA CAG1760 Arg Glu Arg Leu Lys Gln Arg Gln Gln Leu Phe Asp Ser Gln Gln 560 565570 GGA TGG TTT GAA GGA TGG TTC AAC AAG TCC CCC TGG TTT ACA ACC 1805 GlyTrp Phe Glu Gly Trp Phe Asn Lys Ser Pro Trp Phe Thr Thr 575 580 585 CTAATT TCC TCC ATT ATG GGC CCC TTA CTA ATC CTA CTC CTA ATT 1850 Leu Ile SerSer Ile Met Gly Pro Leu Leu Ile Leu Leu Leu Ile 590 595 600 CTC CTC TTCGGC CCA TGC ATC CTT AAC CGA TTA GTA CAA TTC GTA 1895 Leu Leu Phe Gly ProCys Ile Leu Asn Arg Leu Val Gln Phe Val 605 610 615 AAA GAC AGA ATA TCTGTG GTA CAG GCT TTA ATT TTA ACC CAA CAG 1940 Lys Asp Arg Ile Ser Val ValGln Ala Leu Ile Leu Thr Gln Gln 620 625 630 TAC CAA CAG ATA AAG CAA TACGAT CCG GAC CGA CCA TGA 1979 Tyr Gln Gln Ile Lys Gln Tyr Asp Pro Asp ArgPro 635 640

What is claimed is:
 1. A multivalent, recombinant raccoon poxvirus whichcan infect and replicate in feline cells, and contains more than oneexogenous gene inserted into a region consisting of a hemagglutinin geneof the raccoon poxvirus genome which is non-essential for viralreplication, wherein: (a) the exogenous genes are operably linked to apromoter for expression; and (b) each exogenous gene encodes a felinepathogen antigen.
 2. The multivalent recombinant raccoon poxvirusaccording to claim 1, wherein the exogenous genes encode feline pathogenantigens selected from the group consisting of FELV Env, FIV Gag, FIVEnv, FIPV M, FIPV N, FCV capsid protein, FPV VP2, and rabies-G.
 3. Themultivalent recombinant raccoon poxvirus according to claim 1, whereinthe exogenous genes are inserted as an expression cassette.
 4. Themultivalent recombinant raccoon poxvirus according to claim 1, whereinthe recombinant raccoon poxvirus is produced by a recombination processcomprising the steps of: (a) inserting more than one exogenous gene intoan insertion vector which has sequences, flanking the inserted genes,having sufficient homology to the region of the raccoon poxvirus genometo promote recombination of the inserted genes into the hemagglutiningene; (b) introducing both the insertion vector containing the exogenousgenes, and raccoon poxvirus into susceptible host cells; and (c)selecting the recombinant raccoon poxvirus, containing the exogenousgenes which have recombined into the raccoon poxvirus genome, fromplaques resulting from step (b).
 5. The multivalent recombinant raccoonpoxvirus according to claim 4, wherein the exogenous genes are insertedas an expression cassette.
 6. The multivalent recombinant raccoonpoxvirus according to claim 1, wherein the exogenous genes consist oftwo or more genes selected from the group consisting of a FCV capsidprotein gene, a FPV VP2 gene, and rabies-G gene.
 7. The multivalentrecombinant raccoon poxvirus according to claim 6, wherein the exogenousgenes are inserted as an expression cassette.
 8. The multivalentrecombinant raccoon poxvirus according to claim 6, wherein therecombinant raccoon poxvirus is produced by a recombination processcomprising the steps of: (a) inserting two or more genes selected fromthe group consisting of FCV capsid protein gene, FPV VP2 gene and therabies-G gene into an insertion vector which has sequences, flanking theinserted genes, having sufficient homology to a hemagglutinin gene inthe raccoon poxvirus genome to promote recombination of the insertedgenes into the hemagglutinin gene; (b) introducing both the insertionvector containing the inserted genes, and raccoon poxvirus intosusceptible host cells; and (c) selecting the recombinant raccoonpoxvirus, containing the FPV VP2 gene and the rabies-G gene which haverecombined into the raccoon poxvirus genome, from plaques resulting fromstep (b).
 9. The multivalent recombinant raccoon poxvirus according toclaim 8, wherein the sequences flanking the inserted genes consistessentially of a left arm consisting of SEQ ID NO:8, and a right armconsisting of SEQ ID NO:9.
 10. The multivalent recombinant raccoonpoxvirus according to claim 8, wherein the exogenous genes are insertedas an expression cassete.
 11. A method of making a multivalentrecombinant raccoon poxvirus, which can infect and replicate in felinecells, by a recombination process comprising the steps of: (a) insertingmore than one exogenous gene into an insertion vector which hassequences, flanking the inserted genes, having sufficient homology to aregion consisting of a hemagglutinin gene of the raccoon poxvirus genometo promote recombination of the inserted genes into the region; (b)introducing both the insertion vector containing the exogenous genes,and raccoon poxvirus into susceptible host cells; and (c) selecting therecombinant raccoon poxvirus, containing the exogenous genes which haverecombined into the raccoon poxvirus genome, from plaques resulting fromstep (b).
 12. The method according to claim 11, wherein the exogenousgenes are each operably linked to a promoter, and encode feline pathogenantigens selected from the group consisting of FeLV Env, FIV Gag, FIVEnv, FIPV M, FIPV N, FCV capsid protein, FPV VP2, and rabies-G.
 13. Amultivalent recombinant raccoon poxvirus, which can infect and replicatein feline cells, containing more than one exogenous gene inserted intotwo regions of the raccoon poxvirus genome which are non-essential forviral replication, wherein: (a) the exogenous genes are operably linkedto a promoter for expression; (b) each exogenous gene encodes a felinepathogen antigen; and (c) at least one exogenous gene is inserted into athymidine kinase gene, and at least one exogenous gene is inserted intoa hemagglutinin gene.
 14. The multivalent recombinant raccoon poxvirusaccording to claim 13, wherein the exogenous genes encode felinepathogen antigens selected from the group consisting of FELV Env, FIVGag, FIV Env, FIPV M, FIPV N, FCV capsid protein, FPV VP2, and rabies-G.15. The multivalent recombinant raccoon poxvirus according to claim 13,wherein the exogenous genes are inserted as an expression cassette. 16.The multivalent recombinant raccoon poxvirus according to claim 13,wherein the recombinant raccoon poxvirus is produced by a recombinationprocess comprising the steps of: (a) inserting at least one exogenousgene into an insertion vector which has sequences, flanking the insertedgenes, having sufficient homology to the thymidine kinase gene topromote recombination of the inserted genes into the thymidine kinasegene region; (b) inserting at least one exogenous gene into an insertionvector which has sequences, flanking the inserted genes, havingsufficient homology to the hemagglutinin gene to promote recombinationof the inserted genes into the hemagglutinin gene region; (c)introducing both the insertion vector containing the exogenous genes,and raccoon poxvirus into susceptible host cells; and (d) selecting therecombinant raccoon poxvirus, containing the exogenous genes which haverecombined into the raccoon poxvirus genome, from plaques resulting fromstep (b).
 17. The multivalent recombinant raccoon poxvirus according toclaim 16, wherein the exogenous genes are inserted as an expressioncassette.
 18. The multivalent recombinant raccoon poxvirus according toclaim 13, wherein the exogenous genes consist of a gene that encodes anFPV VP2 protein, and a gei-L tbht encodes rabies-G.
 19. The multivalentrecombinant raccoon poxvirus according to claim 18, wherein theexogenous genes are inserted as an expression cassette.
 20. A method ofmaking a multivalent recombinant raccoon poxvirus, which can infect andreplicate in feline cells, by a recombination process comprising thesteps of: (a) inserting at least one exogenous gene into an insertionvector which has sequences, flanking the inserted genes, havingsufficient homology to the thymidine kinase gene of the raccoon poxvirusgenome to promote recombination of the inserted genes into the thymidinekinase gene region; (b) inserting at least one exogenous gene into aninsertion vector which has sequences, flanking the inserted genes,having sufficient homology to the hemagglutinin gene to promoterecombination of the inserted genes into the hemagglutinin gene region;(c) introducing both the insertion vector containing the exogenousgenes, and raccoon poxvirus into susceptible host cells; and (d)selecting the recombinant raccoon poxvirus, containing the exogenousgenes which have recombined into the raccoon poxvirus genome, fromplaques resulting from step (b).
 21. The method according to claim 20,wherein the exogenous genes are each operably linked to a promoter, andencode feline pathogen antigens selected from the group consisting ofFeLV Env, FIV Gag, FIV Env, FIPV M, FIPV N, FCV capsid protein, FPV VP2,and rabies-G.